Direct binding assay for identifying inhibitors of HCV polymerase

ABSTRACT

A method for identifying compounds binding to HCV polymerase comprising the steps of: contacting said HCV polymerase or an analog thereof with a probe formula I: 
                         
wherein A is O, S, N, NR 1 , or CR 1 , wherein R 1  is defined herein;
     ----- represents either a single or a double bond;   R 2  is selected from: H, halogen, R 21 , OR 21 , SR 21 , COOR 21 , SO 2 N(R 22 ) 2 , N(R 22 ) 2 , CON(R 22 ) 2 , NR 22 C(O)R 22  or NR 22 C(O)NR 22  wherein R 21  and each R 22  is defined herein;   B is NR 3  or CR 3 , wherein R 3  is defined herein;   with the proviso that, when A is not N, then one of A or B is either CR 1  or CR 3 ,   K is N or CR 4 , wherein R 4  is defined herein;   L is N or CR 5 , wherein R 5  has the same definition as R 4  defined above;   M is N or CR 7 , wherein R 7  has the same definition as R 4  defined above;   R 5  is C(Y 1 )Z wherein Y 1  is O or S; and   Z is N(R 6a )R 6  or OR 6 , wherein R 6a  is H or alkyl or NR 61 R 62  wherein R 61  and R 62  are defined herein; and R 6  is H, alkyl, cycloalkyl, alkenyl, Het, alkyl-aryl, alkyl-Het; or R 6  is   
                         
wherein R 7  and R 8  and Q are as defined herein;
     Y 2  is O or S;   R 9  is H, (C 1-6  alkyl), (C 3-7 )cycloalkyl or (C 1-6 )alkyl-(C 3-7 )cycloalkyl, aryl, Het, (C 1-6 )alkyl-aryl or (C 1-6 )alkyl-Het, all of which optionally substituted with R 90 ; or R 9  is covalently bonded to either of R 7  or R 8  to form a 5- or 6-membered heterocycle; or a salt thereof; where the probe comprises a detectable label attached to any suitable position, whereby said probe binds to an HCV polymerase or an analog thereof and is capable of being displaced by an inhibitor thereof.

RELATED APPLICATIONS

Benefit of U.S. Provisional Application Ser. No. 60/310,272 filed Aug. 7, 2001, is hereby claimed and said application is incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates generally to a method for identifying inhibitors of the HCV RNA dependent RNA polymerase. Particularly, this method uses a novel probe in a competitive assay to identify HCV polymerase inhibitors and determine their potency. More particularly, this invention relates to the use of a probe which binds with specificity to the polymerase, and which is capable of being displaced by inhibitors of the enzyme.

BACKGROUND OF THE INVENTION

Hepatitis C virus (HCV) is the major etiological agent of post-transfusion and community-acquired non-A non-B hepatitis worldwide. It is estimated that over 200 million people worldwide are infected by the virus. A high percentage of carriers become chronically infected and many progress to chronic liver disease, so called chronic hepatitis C. This group is in turn at high risk for serious liver disease such as liver cirrhosis, hepatocellular carcinoma and terminal liver disease leading to death.

The mechanism by which HCV establishes viral persistence and causes a high rate of chronic liver disease has not been thoroughly elucidated. It is not known how HCV interacts with and evades the host immune system.

HCV is an enveloped positive strand RNA virus in the Flaviviridae family. The single strand HCV RNA genome is of positive polarity and comprises one open reading frame (ORF) of approximately 9600 nucleotides in length, which encodes a linear polyprotein of approx. 3010 amino acids. In infected cells, this polyprotein is cleaved at multiple sites by cellular and viral proteases to produce structural and non-structural (NS) proteins. The structural proteins (C, E1, E2 and E2-p7) comprise polypeptides that constitute the virus particle (Hijikata, M. et al., 1991, Proc. Natl. Acad. Sci. USA. 88, 5547-5551; Grakoui, A. et al., 1993(a), J. Virol. 67,1385-1395). The non-structural proteins (NS2, NS3, NS4A, NS4B, NS5A, NS5B) encode for enzymes or accessory factors that catalyze and regulate the replication of the HCV RNA genome. Processing of the structural proteins is catalyzed by host cell proteases (Hijikata et al., 1991, supra). The generation of the mature non-structural proteins is catalyzed by two virally encoded proteases. The first is the NS2/3 zinc-dependent metalloprotease which auto-catalyses the release of the NS3 protein from the polyprotein. The released NS3 contains a N-terminal serine protease domain (Grakoui A, et al., 1993(b), Proc Natl Acad Sci USA, 90, 10583-7; Hijikata, M. et al., 1993, J. Virol. 67, 4665-4675.) and catalyzes the remaining cleavages from the polyprotein. The released NS4A protein has at least two roles. First, forming a stable complex with NS3 protein and assisting in the membrane localization of the NS3/NS4A complex (Kim et al., Arch Virol. 1999, 144: 329-343) and second, acting as a cofactor for NS3 protease activity. This membrane-associated complex, in turn catalyzes the cleavage of the remaining sites on the polyprotein, thus effecting the release of NS4B, NS5A and NS5B (Bartenschlager, R. et al., 1993, J. Virol., 67, 3835-3844; Grakoui et al., 1993(a) supra; Hijikata et al., 1993 supra; Love, R. A. et al., 1996, Cell, 87, 331-342; reviewed in Kwong AD. et al., 1998, Antiviral Res., 40, 1-18). The C-terminal segment of the NS3 protein also harbors nucleoside triphosphatase and RNA helicase activity (Kim, D. W. et al., 1995, Biochem. Biophys. Res. Comm., 215,160-166). The function of the protein NS4B is unknown. NS5A, a highly phosphorylated protein, seems to be responsible for the Interferon resistance of various HCV genotypes (Gale Jr. et al. 1997 Virology 230, 217; Reed et al., 1997, J. Virol. 71, 7187). NS5B is an RNA-dependent RNA polymerase (RdRp) that is involved in the replication of HCV.

To better understand the mechanism of HCV RNA replication and to develop appropriate in vitro systems, biochemical analyses of the NS5B protein have been performed. Full-length NS5B has been produced and purified as a non-fusion protein from insect cells infected with recombinant baculovirus (S. -E. Behrens et al., 1996, EMBO J., 15:12-22; R. de Francesco et al, 1996, Methods Enzymol., 275:58-67) or as a tagged protein from both insect cells (V. Lohmann et al., 1997, J. Virol., 71:8416-8428; V. Lohmann et al., 1998, Virology 249:108-118) and E. coli (Z. -H. Yuan et al, 1997, BBRC 232:231-235). In vitro, the RdRp activity of recombinant NS5B is dependent on an RNA template and requires RNA or DNA as a primer (S. -E. Behrens et al, 1996, EMBO J. 15:12-22; V. Lohmann et al., 1997, J. Virol., 71:8416-8428). On RNA templates of heteropolymeric sequences, the 3′-OH of the template is used as a primer and elongation proceeds via a “snap-back” mechanism, leading to a double-stranded molecule in which template and product RNA are covalently linked (S. -E. Behrens et a., 1996, EMBO J., 15:12-22; V. Lohman et al., 1998, Virology, 249:108-118; G. Luo et al., 2000, J. Virol. 74:851-863). Recently, several groups also demonstrated that the HCV NS5B protein is able to initiate RNA synthesis de novo (J. Oh et al, 1999, J. Virol. 73:7694-7702; X. Sun et al., 2000, BBRC 268:798-803; W. Zhong et al., 2000, J. Virol. 74:2017-2022).

The NS5B RdRp has been crystallized to reveal a structure reminiscent of other nucleic acid polymerases (S. Bressanelli et al., 1999, PNAS USA 96:13034-13039; H. Ago et al., 1999, Structure 7:1417-1426; C. A. Lesburg et al, 1999, Nature Struct. Biol., 6:937-943). A comprehensive understanding of the differences between HCV and cellular polymerases will facilitate the design of specific inhibitors of HCV replication. Detailed kinetic information will also help in understanding the molecular basis of HCV NS5B-catalyzed nucleotide incorporation and subsequently the mechanistic characterization of the inhibitors.

Previous studies (S. -E. Behrens et a., 1996, EMBO J. 15:12-22; R. de Francesco et al., 1996, Methods Enzymol. 275:58-67; V. Lohmann et al., 1997, J. Virol. 71:8416-8428; V. Lohmann et al., 1998, Virology 249:108-118) provided little information with regard to the proportion of the polymerase RNA complexes that are competent for catalysis. Some recent studies investigated the template and primer requirements for HCV NS5B-directed RNA replication. Templates with 3′-termini free of secondary structures and short primers 2 or 3 nucleotides (nt) long were preferred for efficient initiation of RNA synthesis (W. Zhong et a., 2000, J. Virol. 74:9134-9143). In de novo initiation of RNA synthesis, however, NS5B needs a template with a stable secondary structure and a single-stranded sequence that contains at least one 3′-cytidylate.

Viral polymerases represent attractive targets for therapeutic inhibition of viral replication. The discovery of new antiviral agents often involves screening of large numbers of samples for inhibition of the target activity using either in vitro or in vivo assays. In general, polymerases are assayed by monitoring the incorporation of either ³H—, α-³²P or α-³³P-labeled mononucleotides into oligonucleotide products, or by the extension of 5′-end-labeled primers. Products incorporated into the extended primers are captured or separated using common filter assays, acid precipitation, or denaturing gel electrophoresis.

The HCV NS5B polymerase is a prime target in the search for inhibitors of HCV replication. Different preparations of the HCV polymerase exhibit varying efficiencies of product formation with a variety of RNA substrates. Moreover, the activity of purified recombinant NS5B polymerase varies significantly with specific RNA substrates. In addition, the in vitro RNA polymerase activity of NS5B is extremely sensitive to ionic strength, and salt concentrations exceeding 100 mM inhibit the reaction. Hence the ability to determine the potency of inhibitors at various salt concentrations is restricted by this limitation of standard enzymatic reactions. Also, HCV polymerase enzymatic assays disclosed in the prior art provide IC₅₀ values as representative measurements of inhibitor potencies. For inhibitors that are competitive with either RNA or NTP, the IC₅₀ value is proportional to the concentration of substrates in the assay and will vary depending on the concentration of these components.

In an effort to overcome the limitations of HCV polymerase assays that use sub-optimal and poorly characterized RNA substrates, the Applicants have developed an assay for identifying specific inhibitors of the HCV polymerase that is independent of RNA.

It is therefore an advantage of the present invention to provide an assay that permits a direct measurement of inhibitor potencies (reflected by K_(d) values as an unequivocal determination of inhibitor potency) under defined conditions, irrespective of the substrate concentration.

The direct binding assay of this invention is amenable to adjustments in salt concentration or pH levels beyond the restricted range required for RNA polymerization. This type of assay is amenable to a high sensitivity and a high throughput format.

It is a further advantage of the present invention to provide a probe that binds to the polymerase with a high affinity, and which is displaced by inhibitors of the enzyme.

It is a further advantage to provide an assay that is applicable to HCV polymerases of different genotypes.

SUMMARY OF THE INVENTION

In a first aspect of the invention, there is provided a method for identifying compounds binding to HCV polymerase comprising the steps of:

-   -   a) contacting an HCV polymerase or an analog thereof with a         probe being capable of binding to an HCV polymerase or an analog         thereof, said probe being displaceable by an inhibitor thereof,         so as to form a complex comprising said probe bound to said         polymerase;     -   b) measuring a signal emitted from said probe in said complex to         establish a base line level;     -   c) incubating the product of step a) with a test compound; and     -   d) measuring the signal from said complex; and     -   e) comparing the signal from step d) with the signal from step         b);         whereby a modulation in said signal is an indication that said         test compound binds to said polymerase.

In a preferred aspect of the first embodiment, the probe is selected from: an isomer, enantiomer, diastereoisomer, or tautomer of a probe represented by formula I:

wherein:

-   A is O, S, N, NR¹, or CR¹, wherein R¹ is selected from the group     consisting of: H, (C₁₋₆)alkyl optionally substituted with:     -   halogen, OR¹¹, SR¹¹ or N(R¹²)₂, wherein R¹¹ and each R¹² is         independently H, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl,         (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-aryl or (C₁₋₆)         alkyl-Het, said aryl or Het optionally substituted with R¹⁰; or     -   both R¹² are covalently bonded together and to the nitrogen to         which they are both attached to form a 5, 6 or 7-membered         saturated heterocycle; -   ----- represents either a single or a double bond; -   R² is selected from: H, halogen, R²¹, OR²¹, SR²¹, COOR²¹,     SO₂₁N(R²²)₂, N(R²²)₂, CON(R²²)₂, NR²²C(O)R²² or NR²²C(O)NR²² wherein     R²¹ and each R²² is independently H, (C₁₋₆)alkyl, haloalkyl,     (C₂₋₆)alkenyl, (C₃₋₇)cycloalkyl, (C₂₋₆)alkynyl, (C₅₋₇)cycloalkenyl,     6 or 10-membered aryl or Het, said R²¹ and R²² being optionally     substituted with R²⁰; -   or both R²² are bonded together to form a 5, 6 or 7-membered     saturated heterocycle with the nitrogen to which they are attached; -   B is NR³ or CR³, wherein R³ is selected from (C₁₋₆)alkyl, haloalkyl,     (C₃₋₇)cycloalkyl,     -   (C₆₋₁₀)bicycloalkyl, 6- or 10-membered aryl, Het,         (C₁₋₆)alkyl-aryl or (C₁₋₆)alkyl-Het, said alkyl, cycloalkyl,         bicycloalkyl, aryl, Het, alkyl-aryl and alkyl-Het being         optionally substituted with from 1 to 4 substituents selected         from: halogen, or         -   a) (C₁₋₆)alkyl optionally substituted with:             -   —OR³¹ or SR³¹ wherein R³¹ is H, (C₁₋₆alkyl),                 (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl,                 Het, (C₁₋₆)alkyl-aryl or (C₁₋₆) alkyl-Het; or             -   —N(R³²)₂ wherein each R³² is independently H,                 (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl,                 (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het,                 (C₁₋₆)alkyl-aryl or (C₁₋₆)alkyl-Het; or both R³² are                 covalently bonded together and to the nitrogen to which                 they are attached to form a 5, 6 or 7-membered saturated                 heterocycle;         -   b) OR³³ wherein R³³ is H, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl or             (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆)alkyl-aryl or             (C₁₋₆)alkyl-Het;         -   c) SR³⁴ wherein R³⁴ is H, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, or             (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆)alkyl-aryl or             (C₁₋₆)alkyl-Het; and         -   d) N(R³⁵)₂ wherein each R³⁵ is independently H, (C₁₋₆)alkyl,             (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het,             (C₁₋₆)alkyl-aryl or (C₁₋₆)alkyl-Het; or both R³⁵ are             covalently bonded together and to the nitrogen to which they             are attached to form a 5, 6 or 7-membered saturated             heterocycle; -   with the proviso that, when A is not N, then one of A or B is either     CR¹ or CR³; -   K is N or CR⁴, wherein R⁴ is H, halogen, (C₁₋₆)alkyl, haloalkyl,     (C₃₋₇)cycloalkyl or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl; or R⁴ is OR⁴¹ or     SR⁴, COR⁴¹ or NR⁴ COR⁴¹ wherein each R⁴¹ is independently H,     (C₁₋₆)alkyl), (C₃₋₇)cycloalkyl or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl; or     R⁴ is NR⁴²R⁴³ wherein R⁴² and R⁴³ are each independently H,     (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, or both     R⁴² and R⁴³ are covalently bonded together and to the nitrogen to     which they are attached to form a 5, 6 or 7-membered saturated     heterocycle; -   L is N or CR⁵, wherein R⁵ has the same definition as R⁴ defined     above; -   M is N or CR⁷, wherein R⁷ has the same definition as R⁴ defined     above; -   R⁵ is C(Y¹)-Z wherein Y¹ is O or S; -   Z is N(R^(6a))R⁶ or OR⁶, wherein R^(6a) is H or (C₁₋₆)alkyl or     NR⁶¹R⁶² wherein R⁶¹ and R⁶² are each independently H, (C₁₋₆)alkyl,     (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, or both R⁶¹ and R⁶²     are covalently bonded together and to the nitrogen to which they are     both attached to form a 5, 6 or 7-membered saturated heterocycle; or     R⁶² is COOR⁶³ wherein R⁶³ is (C₁₋₆)alkyl, (C₃₋₆)cycloalkyl, said     alkyl or cycloalkyl being optionally substituted with 6- or     10-membered aryl or Het; or R⁶² is COR⁶⁴ wherein R⁶⁴ is C₁₋₆)alkyl,     (C₃₋₆)cycloalkyl -6-or 10-membered aryl or Het; and -   R⁶ is selected from the group consisting of: H, (C₁₋₆)alkyl,     (C₃₋₆)cycloalkyl, (C₂₋₆)alkenyl, 6- or 10-membered aryl, Het,     (C₁₋₆)alkyl-aryl, (C₁₋₆)alkyl-Het, wherein said alkyl, cycloalkyl,     alkenyl, aryl, Het, alkyl-aryl, or alkyl-Het, are all optionally     substituted with R⁶⁰ ; -   or R⁶ is

wherein R¹ and R⁸ are each independently H, (C₁₋₆)alkyl, haloalkyl, (C₃₋₇)cycloalkyl, 6- or 10-membered aryl, Het, (C₁₋₆)alkyl-aryl, (C₁₋₆)alkyl-Het, wherein said alkyl, cycloalkyl, aryl, Het, (C₁₋₆)alkyl-aryl, (C₁₋₆)alkyl-Het are optionally substituted with R⁷⁰; or

-   R⁷ and R⁸ are covalently bonded together to form second     (C₃₋₇)cycloalkyl or a 4, 5- or 6-membered heterocycle having from 1     to 4 heteroatom selected from O, N, and S; or when Z is N(R^(6a))R⁶,     either of R⁷ or R⁸ is covalently bonded to R^(6a) to form a     nitrogen-containing 5-or 6-membered heterocycle; -   Y² is O or S; -   R⁹ is H, (C₁₋₆alkyl), (C₃₋₇)cycloalkyl or     (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆)alkyl-aryl or     (C₁₋₆)alkyl-Het, all of which optionally substituted with R⁹⁰; or -   R⁹ is covalently bonded to either of R⁷ or R⁸ to form a 5- or     6-membered heterocycle; -   Q is a 6- or 10-membered aryl, Het, (C₁₋₆)alkyl-CONH-aryl or     (C₁₋₆)alkyl-CONH-Het, all of which being optionally substituted     with:

or a salt or a derivative thereof;

-   wherein Het is defined as 5- or 6-membered heterocycle having 1 to 4     heteroatoms selected from O, N, and S, or a 9- or 10-membered     heterobicycle having 1 to 4 heteroatoms selected from O, N and S;     and -   R¹⁰, R²⁰ R⁶⁰, R⁷⁰, R⁹⁰ and R¹⁰⁰ is each defined as:     -   1 to 4 substituents selected from: halogen, OPO₃H, NO₂, cyano,         azido, C(═NH)NH₂, C(═NH)NH(C₁₋₆)alkyl or C(═NH)NHCO(C₁₋₆)alkyl;         or     -   1 to 4 substituents selected from:         -   a) (C₁₋₆)alkyl or haloalkyl, (C₃₋₇)cycloalkyl, C₃₋₇             spirocycloalkyl optionally containing 1 or 2 heteroatom,             (C₂₋₆)alkenyl, (C₂₋₈)alkynyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl,             all of which optionally substituted with R¹⁵⁰;         -   b) OR¹⁰⁴ wherein R¹⁰⁴ is H, (C₁₋₆alkyl), (C₃₋₇)cycloalkyl,             or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl             or (C₁₋₆alkyl)Het, said alkyl, cycloalkyl, aryl, Het,             (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het being optionally             substituted with R¹⁵⁰;         -   c) OCOR¹⁰⁵ wherein R¹⁰⁵ is (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl,             (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, Het, (C₁₋₆alkyl)aryl or             (C₁₋₆alkyl)Het, said alkyl, cycloalkyl, aryl, Het,             (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het being optionally             substituted with R¹⁵⁰;         -   d) SR¹⁰⁸, SO₃H, SO₂N(R¹⁰⁸)₂ or SO₂N(R¹⁰⁸)C(O)R¹⁰⁸ wherein             each R¹⁰⁸ is independently H, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl             or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl             or (C₁₋₆alkyl)Het or both R¹⁰⁸ are covalently bonded             together and to the nitrogen to which they are attached to             form a 5, 6 or 7-membered saturated heterocycle, said alkyl,             cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het or             heterocycle being optionally substituted with R¹⁵⁰;         -   e) NR¹¹¹R¹¹² wherein R¹¹¹ is H, (C₁₋₆)alkyl,             (C₃₋₇)cycloalkyl or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het,             (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, and R¹¹² is H, CN,             (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl or             (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl,             (C₁₋₆alkyl)Het, COOR¹¹⁵ or SO₂R¹¹⁵ wherein R¹¹⁵ is             (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, or             (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or             (C₁₋₆alkyl)Het, or both R¹¹¹ and R¹¹² are covalently bonded             together and to the nitrogen to which they are attached to             form a 5, 6 or 7-membered saturated heterocycle, said alkyl,             cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, or             heterocycle being optionally substituted with R¹⁵⁰;         -   f) NR¹¹⁶COR¹¹⁷ wherein R¹¹⁶ and R¹¹7 is each H, (C₁₋₆)alkyl,             (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het,             (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, said (C₁₋₆)alkyl,             (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het,             (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het being optionally             substituted with R¹⁵⁰;         -   g) NR¹¹⁸CONR¹¹⁹R¹²⁰, wherein R¹¹⁸, R¹¹⁹ and R¹²⁰ is each H,             (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl,             aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, or R¹¹⁸ is             covalently bonded to R¹¹⁹ and to the nitrogen to which they             are attached to form a 5, 6 or 7-membered saturated             heterocycle; or R¹¹⁹ and R¹²⁰ are covalently bonded together             and to the nitrogen to which they are attached to form a 5,             6 or 7-membered saturated heterocycle; said alkyl,             cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het,             (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het or heterocycle being             optionally substituted with R¹⁵⁰;         -   h) NR¹²¹COCOR¹²² wherein R¹²¹ and R¹²² is each is H,             (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl,             a 6- or 10-membered aryl, Het, (C₁₋₆alkyl)aryl or             (C₁₋₆alkyl)Het, said alkyl, cycloalkyl, alkyl-cycloalkyl,             aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het being             optionally substituted with R¹⁵⁰; or R¹²² is OR¹²³ or             N(R¹²⁴)₂ wherein R¹²³ and each R¹²⁴ is independently H,             (C₁₋₆alkyl), (C₃₋₇)cycloalkyl, or             (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or             (C₁₋₆alkyl)Het, or R¹²⁴ is OH or O(C₁₋₆alkyl) or both R¹²⁴             are covalently bonded together to form a 5, 6 or 7-membered             saturated heterocycle, said alkyl, cycloalkyl,             alkyl-cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or             (C₁₋₆alkyl)Het and heterocycle being optionally substituted             with R¹⁵⁰;         -   i) COR¹²⁷ wherein R¹²⁷ is H, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl             or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl             or (C₁₋₆alkyl)Het, said alkyl, cycloalkyl, aryl, Het,             (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het being optionally             substituted with R¹⁵⁰;         -   j) COOR¹²⁸ wherein R¹²⁸ is H, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl,             or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl             or (C₁₋₆alkyl)Het, said (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, or             (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl and             (C₁₋₆alkyl)Het being optionally substituted with R¹⁵⁰;         -   k) CONR¹²⁹R¹³⁰ wherein R¹²⁹ and R¹³⁰ are independently H,             (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl,             aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, or both R¹²⁹             and R¹³⁰ are covalently bonded together and to the nitrogen             to which they are attached to form a 5, 6 or 7-membered             saturated heterocycle, said alkyl, cycloalkyl,             alkyl-cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl, (C₁₋₆alkyl)Het             and heterocycle being optionally substituted with R¹⁵⁰;         -   l) aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, all of             which being optionally substituted with R¹⁵⁰; and             -   wherein R¹⁵⁰ is defined as: —1 to 3 substituents                 selected from: halogen, OPO₃H, NO₂, cyano, azido,                 C(═NH)NH₂, C(═NH)NH(C₁₋₆)alkyl or                 C(═NH)NHCO(C₁₋₁₆)alkyl; or     -   1 to 3 substituents selected from:         -   a) (C₁₋₆)alkyl or haloalkyl, (C₃₋₇)cycloalkyl, C₃₋₇             spirocycloalkyl optionally containing 1 or 2 heteroatom,             (C₂₋₆)alkenyl, (C₂₋₈)alkynyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl,             all of which optionally substituted with R¹⁶⁰;         -   b) OR¹⁰⁴ wherein R¹⁰⁴ is H, (C₁₋₆alkyl), (C₃₋₇)cycloalkyl,             or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl             or (C₁₋₆alkyl)Het, said alkyl, cycloalkyl, aryl, Het,             (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het being optionally             substituted with R¹⁶⁰;         -   c) OCOR¹⁰⁵ wherein R¹⁰⁵ is (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl,             (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, Het, (C₁₋₆alkyl)aryl or             (C₁₋₆alkyl)Het, said alkyl, cycloalkyl, aryl, Het,             (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het being optionally             substituted with R¹⁶⁰;         -   d) SR¹⁰⁸, SO₃H, SO₂N(R¹⁰⁸)₂ or SO₂N(R¹⁰⁸)C(O)R¹⁰⁸ wherein             each R¹⁰⁸ is independently H, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl             or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl             or (C₁₋₆alkyl)Het or both R¹⁰⁸ are covalently bonded             together and to the nitrogen to which they are attached to             form a 5, 6 or 7-membered saturated heterocycle, said alkyl,             cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het or             heterocycle being optionally substituted with R¹⁶⁰;         -   e) NR¹¹¹R¹¹² wherein R¹¹¹ is H, (C₁₋₆)alkyl,             (C₃₋₇)cycloalkyl or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het,             (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, and R¹¹⁵ is H, CN,             (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl or             (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl,             (C₁₋₆alkyl)Het, COOR¹¹⁵ or SO₂R¹¹⁵wherein R¹¹⁵ is             (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, or             (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or             (C₁₋₆alkyl)Het, or both R¹¹¹ and R¹¹² are covalently bonded             together and to the nitrogen to which they are attached to             form a 5, 6 or 7-membered saturated heterocycle, said alkyl,             cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, or             heterocycle being optionally substituted with R¹⁶⁰;         -   f) NR¹¹⁶COR¹¹⁷ wherein R¹¹⁶ and R¹¹⁷ is each H, (C₁₋₆)alkyl,             (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het,             (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, said (C₁₋₆)alkyl,             (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het,             (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het being optionally             substituted with R¹⁶⁰;         -   g) NR¹¹⁸CONR¹¹⁹R¹²⁰, wherein R¹¹⁸, R¹¹⁹ and R¹²⁰ is each H,             (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl,             aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, or R¹¹⁸ is             covalently bonded to R¹¹⁹ and to the nitrogen to which they             are attached to form a 5, 6 or 7-membered saturated             heterocycle; or R¹¹⁹ and R¹²⁰ are covalently bonded together             and to the nitrogen to which they are attached to form a 5,             6 or 7-membered saturated heterocycle; said alkyl,             cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het,             (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het or heterocycle being             optionally substituted with R¹⁶⁰;         -   h) NR¹²¹COCOR¹²² wherein R¹²¹ and R¹²² is each is H,             (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl,             a 6- or 10-membered aryl, Het, (C₁₋₆alkyl)aryl or             (C₁₋₆alkyl)Het, said alkyl, cycloalkyl, alkyl-cycloalkyl,             aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het being             optionally substituted with R¹⁶⁰; or R¹²² is OR¹²³ or             N(R¹²⁴)₂ wherein R¹²³ and each R¹²⁴ is independently H,             (C₁₋₆alkyl), (C₃₋₇)cycloalkyl, or             (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or             (C₁₋₆alkyl)Het, or R¹²⁴ is OH or O(C₁₋₆alkyl) or both R¹²⁴             are covalently bonded together to form a 5, 6 or 7-membered             saturated heterocycle, said alkyl, cycloalkyl,             alkyl-cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or             (C₁₋₆alkyl)Het and heterocycle being optionally substituted             with R¹⁶⁰;         -   i) COR¹²⁷ wherein R¹²⁷ is H, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl             or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl             or (C₁₋₆alkyl)Het, said alkyl, cycloalkyl, aryl, Het,             (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het being optionally             substituted with R¹⁶⁰;         -   j) tetrazole, COOR¹²⁸ wherein R¹²⁸ is H, (C₁₋₆)alkyl,             (C₃₋₇)cycloalkyl, or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl,             Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, said (C₁₋₆)alkyl,             (C₃₋₇)cycloalkyl, or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl,             Het, (C₁₋₆alkyl)aryl and (C₁₋₆alkyl)Het being optionally             substituted with R¹⁶⁰; and         -   k) CONR¹²⁹R¹³⁰ wherein R¹²⁹ and R¹³⁰ are independently H,             (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl,             aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, or both R¹²⁹             and R¹³⁰ are covalently bonded together and to the nitrogen             to which they are attached to form a 5, 6 or 7-membered             saturated heterocycle, said alkyl, cycloalkyl,             alkyl-cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl, (C₁₋₆alkyl)Het             and heterocycle being optionally substituted with R¹⁶⁰;             -   wherein R¹⁶⁰ is defined as 1 or 2 substituents selected                 from: tetrazole, halogen, CN, C₁₋₆alkyl, haloalkyl,                 COOR¹⁶¹, SO₃H, SR¹⁶¹, SO₂R¹⁶¹, OR¹⁶¹, N(R¹⁶²)₂,                 SO₂N(R¹⁶²)₂, or CON(R¹⁶²)₂, wherein R¹⁶¹ and each R¹⁶²                 is independently H, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl or                 (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl; or both R¹⁶² are                 covalently bonded together and to the nitrogen to which                 they are attached to form a 5, 6 or 7-membered saturated                 heterocycle,                 wherein said probe comprises a detectable label attached                 to any suitable position, whereby said probe binds to an                 HCV polymerase or an analog thereof and is capable of                 being displaced by an inhibitor thereof.

According to an alternative of this first embodiment, the probe used for the assay does not comprise a detectable label, and the signal measured is the change in intrinsic fluorescence of the HCV polymerase in the presence and absence of said probe.

According to a second aspect of the invention, there is provided the use of a probe according to formula I in the development of an assay for identifying inhibitors of HCV polymerase.

According to a third aspect of the invention, there is provided a kit for testing compounds potentially binding to HCV polymerase, said kit comprising the probe of formula (I) and instructions on how to use said probe for identifying test compounds binding to said polymerase.

BRIEF DESCRIPTION OF THE FIGURES

Having thus generally described the invention, reference will now be made to the accompanying drawings, showing by way of illustration a preferred embodiment thereof, and in which:

FIG. 1 illustrates the titration of probe (i) with the NS5BΔ21-His polymerase. Standard conditions for the Fluorescence anisotropy analysis are described in Example 3. The determined K_(d) value of probe (i) for this polymerase is 12.6 nM.

FIG. 2 illustrates Z′ evaluation for the Fluorescence Polarization assay. A series of positive and negative controls were tested in the 96-well plate polarization assay, using the standard conditions, to determine the standard deviation (SD) of both controls. The Z′ value was then obtained from the following calculation:

$Z^{\prime} = {1 - \frac{\left( {{3\mspace{14mu}{SD}\mspace{14mu}\text{of~~pos.~~ctrls}} + {3\mspace{14mu}{SD}\mspace{14mu}\text{of~~neg.~~ctrls}}} \right)}{\left( {\text{mean~~pos.~~ctrl} - \text{mean~~neg.~~ctrl}} \right)}}$

FIG. 3 illustrates K_(d) determination for Compounds A and B, using the Fluorescence Polarization assay. Standard conditions of the 96-well plate Polarization assay (see Example 4) were used to determine the K_(d) values of the compounds. K_(d) values obtained for compound A and B are 31 and 41 nM, respectively, with Q_(b)/Q_(f) values of 0.67 and 0.72.

FIG. 4 illustrates K_(d) determination for Compounds C and D, using the Fluorescence Polarization assay. Standard conditions of the 96-well plate Polarization assay (see Example 4) have been used to determine the K_(d) values of some of our compounds. K_(d) values obtained for compound C and D are 231 nM and 1.08 uM, respectively, with Q_(b)/Q_(f) values of 0.74 and 0.66.

FIGS. 5 to 8 illustrate the titration of probe (i) with the NS5BΔ21-His in the presence of increasing (from 30 mM to 200 mM) concentration of NaCl. Standard conditions of the Fluorescence anisotropy analysis are described in Example 3. K_(d) values obtained for this polymerase are 15.3 nM (30 mM NaCl), 39 nM (100 mM NaCl), 78 nM (150 mM NaCl) and finally 122 nM (200 mM NaCl).

FIG. 9 illustrates the titration of probe (i) with the NS5BΔ21-His in Phosphate buffer pH 6.5. Standard conditions of the Fluorescence anisotropy analysis are described in Example 3. The K_(d) of probe (i) for this polymerase under these conditions is 33 nM.

FIG. 10 illustrates the titration of probe (i) with the His-NS5BΔ21 polymerase. Standard conditions of the Fluorescence anisotropy analysis are described in Example 3. The K_(d) of probe (i) for this N-terminally tagged polymerase is 18.1 nM.

FIG. 11 illustrates the titration of probe (ii) with the GBV-BΔ23-His polymerase. Standard conditions of the Fluorescence anisotropy analysis are described in Example 3. The K_(d) of probe (ii) for this distantly related polymerase is 1.79 uM (estimated value with an incomplete curve).

FIG. 12 illustrates the titration of probe (ii) with the His-NS5BΔ21(H77c, HCV genotype 1a) polymerase. Standard conditions of the Fluorescence anisotropy analysis are described in Example 3. The K_(d) of probe (ii) for this HCV genotype 1a polymerase is 18.2 nM.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

The following definitions apply unless otherwise noted:

The term “affinity tag” means a moiety whose strong affinity for a ligand can be used to extract from a solution the entity to which the tag is attached. Examples of such tags include biotin or a derivative thereof, a histidine polypeptide, a polyarginine, an amylose sugar moiety or a defined epitope recognizable by a specific antibody. Such affinity tags are attached to the probe by well-known methods. The corresponding affinity ligands are also well known in the art.

An “analog” of the HCV NS5B polypeptide, for example HCV polymerase or a fragment thereof means a polypeptide modified by varying the amino acid sequence of the protein, e.g. by manipulation of the nucleic acid encoding the protein or by altering the protein itself. Such analogs of the natural amino acid sequence may involve insertion, addition, deletion or substitution of one or more amino acids, and may or may not alter the functional activity of the original HCV NS5B polypeptide. As mentioned above, the HCV NS5B polypeptide or protein used in the assay/method of the invention includes any fragment, derivative, variant or mutant which is derived from a HCV NS5B polypeptide and which retains at least one property or other characteristic of the HCV NS5B polypeptide.

The term “detectable label” refers to any group that is linked to a probe of the present invention such that when the probe is associated with the polymerase target, such label allows recognition either directly or indirectly of the probe such that it can be detected, measured and quantified. Examples of such “detectable labels” are intended to include, but are not limited to: photoreactive groups, fluorescent labels, chemiluminescent labels, colorimetric labels, enzymatic markers, radioactive isotopes. Such labels are attached to the probe by well known methods.

As used herein, the term “linker” refers to a chain of between 1 and 20 atoms selected from the group consisting of C, N, O, and S that covalently connects the aforesaid label to a probe of the present invention. Examples of such a chain include, but are not limited to, the following:

These linkers can also comprise a pair of affinity-tag/affinity-ligand, which together, bind the compound to a detectable label.

The term “photoreactive group” means a group that is transformed, upon activation by light, from an inert group to a reactive species, such as a free radical. Examples of such groups include, but are not limited to, benzophenones, azides, and the like.

As used herein, the term “probe” refer to a compound of formula (I) that is capable of binding to an HCV polymerase in a covalent or non-covalent manner. When the probe is bound in a non-covalent manner, it can be displaced by test compounds. When bound in a covalent manner, the probe can be used for cross-linking experiments wherein the HCV polymerase-probe adduct formation can be quantified and inhibited by test compounds.

As used herein, the terms “(C₁₋₃)alkyl”, “(C₁₋₄)alkyl” or “(C₁₋₆)alkyl”, either alone or in combination with another radical, are intended to mean acyclic straight or branched chain alkyl radicals containing up to three, four and six carbon atoms respectively. Examples of such radicals include methyl, ethyl, propyl, butyl, hexyl, 1-methylethyl, 1-methylpropyl, 2-methylpropyl, 1,1-dimethylethyl.

As used herein, the term “(C₂₋₆) alkenyl”, either alone or in combination with another radical, is intended to mean an unsaturated, acyclic straight chain radical containing two to six carbon atoms.

As used herein, the term (C₂₋₆)alkynyl” either alone or in combination with another group, is intended to mean an unsaturated, acyclic straight chain sp hybridized radical containing 2 to six carbon atoms.

As used herein, the term “(C₃₋₇)cycloalkyl”, either alone or in combination with another radical, means a cycloalkyl radical containing from three to seven carbon atoms and includes cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl.

As used herein, the term “(C₅₋₇)cycloalkenyl”, either alone or in combination with another radical, means an unsaturated cyclic radical containing five to seven carbon atoms.

As used herein, the term “aryl”, or “6- or 10-membered aryl” either alone or in combination with another radical means aromatic radical containing six or ten carbon atoms, for example phenyl or naphthyl.

As used herein, the term “COOH” refers to a carboxylic acid group. It is well known to one skilled in the art that carboxylic acid groups may be substituted by functional group equivalents. Examples of such functional group equivalents that are contemplated by this invention include, but are not limited to, esters, amides, or boronic acids.

As used herein, the term “functional group equivalent” is intended to mean an element or a substituted derivative thereof, that is replaceable by another element that has similar electronic, hybridization or bonding properties.

As used herein, the term “halo” means a halogen atom and includes fluorine, chlorine, bromine and iodine.

As used herein, the term “haloalkyl” is intended to mean an alkyl that is described above in which each hydrogen atom may be successively replaced by a halogen atom, for example CH₂Br or CF₃.

As used herein the term heteroatom means O, S or N.

As used herein, the term “heterocycle”, either alone or in combination with another radical, means a monovalent radical derived by removal of a hydrogen from a five-, six-, or seven-membered saturated or unsaturated (including aromatic) heterocycle containing from one to four heteroatoms selected from nitrogen, oxygen and sulfur. Furthermore, “heterobicyclic” as used herein, means a heterocycle as defined above fused to one or more other cycle, be it a heterocycle or any other cycle. Examples of such heterocycles include, but are not limited to, pyrrolidine, tetrahydrofuran, thiazolidine, pyrrole, thiophene, coumarin, hydantoin, diazepine, 1H-imidazole, isoxazole, thiazole, tetrazole, piperidine, 1 ,4-dioxane, 4-morpholine, pyridine, pyridine-N-oxide, pyrimidine, thiazolo[4,5-b]-pyridine, quinoline, or indole, or the following heterocycles:

As used herein, the term “9- or 10-membered heterobicycle” or “heterobicycle” either alone or in combination with another radical, means a heterocycle as defined above fused to one or more other cycle, be it a heterocycle or any other cycle. Examples of such heterobicycles include, but are not limited to, thiazolo[4,5-b]-pyridine, quinoline, or indole, or the following:

As used herein, the term “Het” defines a 5- or 6-membered heterocycle having 1 to 4 heteroatoms selected from O, N, and S, or a 9- or 10-membered heterobicycle having 1 to 4 heteroatoms selected from O, N and S.

As used herein, the term “OH” refers to a hydroxyl group. It is well known to one skilled in the art that hydroxyl groups may be substituted by functional group equivalents. Examples of such functional group equivalents that are contemplated by this invention include, but are not limited to, ethers, sulfhydryls, and primary, secondary or tertiary amines.

As used herein, the term “SH” refers to a sulfhydryl group. It is intended within the scope of the present invention that, whenever a “SH” or “SR” group is present, it can also be substituted by any other appropriate oxidation state such as SOR, SO₂R, or SO₃R.

It is intended that the term “substituted” when applied in conjunction with a radical having more than one moiety such as C₁₋₆alkyl-aryl, or C₁₋₆alkyl-Het, such substitution applies to both moieties i.e. both the alkyl and aryl or Het moieties can be substituted with the defined substituents.

DESCRIPTION OF PREFERRED EMBODIMENTS

Preferably, according to the first aspect, the invention provide a method for identifying inhibitors of HCV polymerase comprising the steps of:

-   -   a) contacting an HCV polymerase with a probe of formula I so as         to form a complex comprising said probe bound to said         polymerase;     -   b) measuring a signal from said complex to establish a base line         level;     -   c) incubating the product of step a) with a test compound;     -   d) measuring the signal from said complex; and     -   e) comparing the signal from step d) with the signal from step         b);         whereby a decrease in said signal is an indication that said         test compound is an inhibitor of said polymerase.

As will be understood by a person skilled in the art, the association of a specific probe of the invention with the NS5B polymerase can be measured directly or indirectly in a variety of ways. The probe and NS5B polymerase need not be labeled and affinity tagged respectively. The association of a specific probe with the HCV NS5B polymerase can be monitored and quantified directly by a change in the intrinsic spectral properties of a tagged or un-tagged NS5B protein and/or by a change in the intrinsic spectral properties of a specific probe. A direct measurement of inhibitor-NS5B association can also be achieved by immobilizing one of these two components on a matrix and measuring association through plasma-resonance detection technology. An assay that quantifies probe-NS5B complex association may also incorporate a photo-reactive label (such as a phenyl-azide or benzophenone) on the probe (for example probes (v) and (vi) below) and measure the amount of label irreversibly bound to the NS5B (adduct) following photo-activation of the probe.

Preferably, according to a first aspect of the present invention, there is provided a probe of formula:

wherein

-   R¹ is selected from the group consisting of: H or (C₁₋₆)alkyl; -   R² is CON(R²²)₂, wherein each R²² is independently H, (C₁₋₆)alkyl,     (C₃₋₇)cycloalkyl, (C₅₋₇)cycloalkenyl, 6 or 10-membered aryl or Het,     or both R²² are bonded together to form a 5, 6 or 7-membered     saturated heterocycle with the nitrogen to which they are attached; -   or R² is selected from: H, halogen, (C₁₋₆)alkyl, haloalkyl,     (C₂₋₆)alkenyl, (C₅₋₇)cycloalkenyl, 6 or 10-membered aryl or Het;     wherein each of said alkyl, haloalkyl, (C₂₋₆)alkenyl,     (C₅₋₇)cycloalkenyl, aryl or Het is optionally substituted with R²⁰,     wherein R²⁰ is defined as:     -   1 to 4 substituents selected from: halogen, NO₂, cyano, azido,         C(═NH)NH₂, C(═NH)NH(C₁₋₆)alkyl or C(═NH)NHCO(C₁₋₆)alkyl; or     -   1 to 4 substituents selected from:         -   a) (C₁₋₆)alkyl or haloalkyl, (C₃₋₇)cycloalkyl,             (C₂₋₆)alkenyl, (C₂₋₈)alkynyl, (C₁₋₆) alkyl-(C₃₋₇)cycloalkyl,             all of which optionally substituted with R¹⁵⁰;         -   b) OR¹⁰⁴ wherein R¹⁰⁴ is H, (C₁₋₆alkyl), (C₃₋₇)cycloalkyl,             or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl             or (C₁₋₆alkyl)Het, said alkyl, cycloalkyl, aryl, Het,             (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het being optionally             substituted with R¹⁵⁰;         -   c) OCOR¹⁰⁵ wherein R¹⁰⁵ is (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl,             (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, Het, (C₁₋₆alkyl)aryl or             (C₁₋₆alkyl)Het, said alkyl, cycloalkyl, aryl, Het,             (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het being optionally             substituted with R¹⁵⁰;         -   d) SR¹⁰⁸, SO₃H, SO₂N(R¹⁰⁸)₂ or SO₂N(R¹⁰⁸)C(O)R¹⁰⁸ wherein             each R¹⁰⁸ is independently H, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl             or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl             or (C₁₋₆alkyl)Het or both R¹⁰⁸ are covalently bonded             together and to the nitrogen to which they are attached to             form a 5, 6 or 7-membered saturated heterocycle, said alkyl,             cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het or             heterocycle being optionally substituted with R¹⁵⁰;         -   e) NR¹¹¹R¹¹² wherein R¹¹¹ is H, (C₁₋₆)alkyl,             (C₃₋₇)cycloalkyl or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het,             (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, and R¹¹² is H, CN,             (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl or             (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl,             (C₁₋₆alkyl)Het, COOR¹¹⁵ or SO₂R¹¹⁵wherein R¹¹⁵ is             (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, or             (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or             (C₁₋₆alkyl)Het, or both R¹¹¹ and R¹¹² are covalently bonded             together and to the nitrogen to which they are attached to             form a 5, 6 or 7-membered saturated heterocycle, said alkyl,             cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, or             heterocycle being optionally substituted with R¹⁵⁰;         -   f) NR¹¹⁶COR¹¹⁷ wherein R¹¹⁶ and R¹¹⁷ is each H, (C₁₋₆)alkyl,             (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het,             (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, said (C₁₋₆)alkyl,             (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het,             (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het being optionally             substituted with R¹⁵⁰;         -   g) NR¹¹⁸CONR¹¹⁹R¹²⁰, wherein R¹¹⁸, R¹¹⁹ and R¹²⁰ is each H,             (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl,             aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, or R¹¹⁸ is             covalently bonded to R¹¹⁹ and to the nitrogen to which they             are attached to form a 5, 6 or 7-membered saturated             heterocycle; or R¹¹⁹ and R¹²⁰ are covalently bonded together             and to the nitrogen to which they are attached to form a 5,             6 or 7-membered saturated heterocycle; said alkyl,             cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het,             (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het or heterocycle being             optionally substituted with R¹⁵⁰;         -   h) NR¹²¹COCOR¹²² wherein R¹²¹ and R¹²² is each is H,             (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl,             a 6- or 10-membered aryl, Het, (C₁₋₆alkyl)aryl or             (C₁₋₆alkyl)Het, said alkyl, cycloalkyl, alkyl-cycloalkyl,             aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het being             optionally substituted with R¹⁵⁰; or R¹²² is OR¹²³ or             N(R¹²⁴)₂ wherein R¹²³ and each R¹²⁴ is independently H,             (C₁₋₆alkyl), (C₃₋₇)cycloalkyl, or             (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or             (C₁₋₆alkyl)Het, or R¹²⁴ is OH or O(C₁₋₆alkyl) or both R¹²⁴             are covalently bonded together to form a 5, 6 or 7-membered             saturated heterocycle, said alkyl, cycloalkyl,             alkyl-cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or             (C₁₋₆alkyl)Het and heterocycle being optionally substituted             with R¹⁵⁰;         -   i) COR¹²⁷ wherein R¹²⁷ is H, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl             or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆-alkyl)aryl             or (C₁₋₆alkyl)Het, said alkyl, cycloalkyl, aryl, Het,             (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het being optionally             substituted with R¹⁵⁰;         -   j) COOR¹²⁸ wherein R¹²⁸ is H, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl,             or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl             or (C₁₋₆alkyl)Het, said (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, or             (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl and             (C₁₋₆alkyl)Het being optionally substituted with R¹⁵⁰;         -   k) CONR¹²⁹R¹³⁰ wherein R¹²⁹ and R¹³⁰ are independently H,             (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl,             aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, or both R¹²⁹             and R¹³⁰ are covalently bonded together and to the nitrogen             to which they are attached to form a 5, 6 or 7-membered             saturated heterocycle, said alkyl, cycloalkyl,             alkyl-cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl, (C₁₋₆alkyl)Het             and heterocycle being optionally substituted with R¹⁵⁰;         -   l) aryl, Het, (C1-6alkyl)aryl or (C1-6alkyl)Het, all of             which being optionally substituted with R¹⁵⁰;     -   wherein R¹⁵⁰ is preferably:     -   1 to 3 substituents selected from: halogen, NO₂, cyano or azido;         or     -   1 to 3 substituents selected from:         -   a) (C₁₋₆)alkyl or haloalkyl, (C₃₋₇)cycloalkyl,             (C₂₋₆)alkenyl, (C₂₋₈)alkynyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl,             all of which optionally substituted with R^(160;)         -   b) OR¹⁰⁴ wherein R¹⁰⁴ is H, (C₁₋₆alkyl) or (C₃₋₇)cycloalkyl,             said alkyl or cycloalkyl optionally substituted with R¹⁶⁰;         -   d) SR¹⁰⁸, SO₃H, SO₂N(R¹⁰⁸)₂ or SO₂N(R¹⁰⁸)C(O)R¹⁰⁸ wherein             each R¹⁰⁸ is independently H, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl             or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, or both R¹⁰⁸ are             covalently bonded together and to the nitrogen to which they             are attached to form a 5, 6 or 7-membered saturated             heterocycle, said alkyl, cycloalkyl, aryl, Het and             heterocycle being optionally substituted with R¹⁶⁰;         -   e) NR¹¹¹R¹¹² wherein R¹¹¹ is H, (C₁₋₆)alkyl, or             (C₃₋₇)cycloalkyl, and R¹¹² is H, (C₁₋₆)alkyl or             (C₃₋₇)cycloalkyl, COOR¹¹⁵ or SO₂R¹¹⁵ wherein R¹¹⁵ is             (C₁₋₆)alkyl or (C₃₋₇)cycloalkyl, or both R¹¹¹ and R¹¹² are             covalently bonded together and to the nitrogen to which they             are attached to form a 5, 6 or 7-membered saturated             heterocycle, said alkyl, cycloalkyl and heterocycle being             optionally substituted with R¹⁶⁰;         -   f) NR¹¹⁶COR¹¹⁷ wherein R¹²⁶ and R¹¹⁷ is each H, (C₁₋₆)alkyl             or (C₃₋₇)cycloalkyl said (C₁₋₆)alkyl and (C₃₋₇)cycloalkyl             being optionally substituted with R¹⁶⁰;         -   g) NR¹¹⁸CONR¹¹⁹R¹²⁰, wherein R¹¹⁸, R¹¹⁹ and R¹²⁰ is each H,             (C₁₋₆)alkyl or (C₃₋₇)cycloalkyl, or R¹¹⁸ is covalently             bonded to R¹¹⁹ and to the nitrogen to which they are             attached to form a 5, 6 or 7-membered saturated heterocycle;             or R¹¹⁹ and R¹²⁰ are covalently bonded together and to the             nitrogen to which they are attached to form a 5, 6 or             7-membered saturated heterocycle; said alkyl, cycloalkyl,             and heterocycle being optionally substituted with R¹⁶⁰;         -   h) NR¹²¹COCOR¹²² wherein R¹²¹ is H, (C₁₋₆)alkyl or             (C₃₋₄)cycloalkyl, said alkyl and cycloalkyl being optionally             substituted with R¹⁶⁰; or R¹²² is OR¹²³ or N(R¹²⁴)₂ wherein             R¹²³ and each R¹²⁴ is independently H, (C₁₋₆alkyl) or             (C₃₋₇)cycloalkyl, or both R¹²⁴ are covalently bonded             together to form a 5, 6 or 7-membered saturated heterocycle,             said alkyl, cycloalkyl and heterocycle being optionally             substituted with R¹⁶⁰;         -   i) COR¹²⁷ wherein R¹²⁷ is H, (C₁₋₆)alkyl or             (C₃₋₇)cycloalkyl, said alkyl and cycloalkyl being optionally             substituted with R¹⁶⁰;         -   j) COOR¹²⁸ wherein R¹²⁸ is H, (C₁₋₆)alkyl or             (C₃₋₇)cycloalkyl, said (C₁₋₆)alkyl and (C₃₋₇)cycloalkyl             being optionally substituted with R¹⁶⁰; and         -   k) CONR¹²⁹R¹³⁰ wherein R¹²⁹ and R¹³⁰ are independently H,             (C₁₋₆)alkyl or (C₃₋₇)cycloalkyl, or both R¹²⁹ and R¹³⁰ are             covalently bonded together and to the nitrogen to which they             are attached to form a 5, 6 or 7-membered saturated             heterocycle, said alkyl, cycloalkyl and heterocycle being             optionally substituted with R¹⁶⁰;             -   wherein R¹⁶⁰ is defined as 1 or 2 substituents selected                 from: halogen, CN, C₁₋₆alkyl, haloalkyl, COOR¹⁶¹, OR¹⁶¹,                 N(R¹⁶²)₂, SO₂N(R¹⁶²)₂, or CON(R¹⁶²)₂, wherein R¹⁶¹ and                 each R¹⁶² is independently H, (C₁₋₆)alkyl,                 (C₃₋₇)cycloalkyl or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl; or                 both R¹⁶² are covalently bonded together and to the                 nitrogen to which they are attached to form a 5, 6 or                 7-membered saturated heterocycle; -   R³ is selected from (C₃₋₇)cycloalkyl, (C₆₋₁₀)bicycloalkyl, 6- or     10-membered aryl, or Het; -   R⁵ is —C(O)-Z, wherein -   Z is OR⁶ wherein R⁶ is C₁₋₆alkyl substituted with:     -   1 to 4 substituents selected from: OPO₃H, NO₂, cyano, azido,         C(═NH)NH₂, C(═NH)NH(C₁₋₆)alkyl or C(═NH)NHCO(C₁₋₆)alkyl; or     -   1 to 4 substituents selected from:         -   a) (C₁₋₆)alkyl or haloalkyl, (C₃₋₇)cycloalkyl, C₃₋₇             spirocycloalkyl optionally containing 1 or 2 heteroatom,             (C₂₋₆)alkenyl, (C₂₋₈)alkynyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl,             all of which optionally substituted with R¹⁵⁰;         -   b) OR¹⁰⁴ wherein R¹⁰⁴ is (C₁₋₆alkyl) substituted with R¹⁵⁰,             (C₃₋₇)cycloalkyl, or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl,             Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, said cycloalkyl,             aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het being             optionally substituted with R¹⁵⁰;         -   c) OCOR¹⁰⁵ wherein R¹⁰⁵ is (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl,             (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, Het, (C₁₋₆alkyl)aryl or             (C₁₋₆alkyl)Het, said alkyl, cycloalkyl, aryl, Het,             (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het being optionally             substituted with R¹⁵⁰;         -   d) SR¹⁰⁸, SO₃H, SO₂N(R¹⁰⁸)₂ or SO₂N(R¹⁰⁸)C(O)R¹⁰⁸ wherein             each R¹⁰⁸ is independently H, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl             or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl             or (C₁₋₆alkyl)Het or both R¹⁰⁸ are covalently bonded             together and to the nitrogen to which they are attached to             form a 5, 6 or 7-membered saturated heterocycle, said alkyl,             cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het or             heterocycle being optionally substituted with         -   e) NR¹¹¹ R¹¹² wherein R¹¹¹ is (C₁₋₆)alkyl substituted with             R¹⁵⁰, (C₃₋₇)cycloalkyl or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl,             aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, and R¹¹² is             CN, (C₁₋₆)alkyl substituted with R⁵⁰, (C₃₋₇)cycloalkyl or             (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl,             (C₁₋₆alkyl)Het, COOR¹¹⁵ or SO₂R¹¹⁵ wherein R¹¹⁵ is             (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, or             (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or             (C₁₋₆alkyl)Het, or both R¹¹¹ and R¹¹² are covalently bonded             together and to the nitrogen to which they are attached to             form a 5, 6 or 7-membered saturated heterocycle, said             cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, or             heterocycle being optionally substituted with R¹⁵⁰;         -   f) NR¹¹⁶COR¹¹⁷ wherein R¹¹⁶ and R¹¹⁷ is each H, (C₁₋₆)alkyl,             (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het,             (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, said (C₁₋₆)alkyl,             (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het,             (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het being optionally             substituted with R¹⁵⁰;         -   g) NR¹¹⁸CONR¹¹⁹R¹²⁰, wherein R¹¹⁸, R¹¹⁹ and R¹²⁰ is each H,             (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl,             aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, or R¹¹⁸ is             covalently bonded to R¹¹⁹ and to the nitrogen to which they             are attached to form a 5, 6 or 7-membered saturated             heterocycle; or R¹¹⁹ and R¹²⁰ are covalently bonded together             and to the nitrogen to which they are attached to form a 5,             6 or 7-membered saturated heterocycle; said alkyl,             cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het,             (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het or heterocycle being             optionally substituted with R¹⁵⁰;         -   h) NR¹²¹COCOR¹²² wherein R¹²¹ and R¹²² is each is H,             (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl,             a 6- or 10-membered aryl, Het, (C₁₋₆alkyl)aryl or             (C₁₋₆alkyl)Het, said alkyl, cycloalkyl, alkyl-cycloalkyl,             aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het being             optionally substituted with R¹⁵⁰; or R¹²² is OR¹²³ or             N(R¹²⁴)₂ wherein R¹²³ and each R¹²⁴ is independently H,             (C₁₋₆alkyl), (C₃₋₇)cycloalkyl, or             (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or             (C₁₋₆alkyl)Het, or R¹²⁴ is OH or O(C₁₋₆alkyl) or both R¹²⁴             are covalently bonded together to form a 5, 6 or 7-membered             saturated heterocycle, said alkyl, cycloalkyl,             alkyl-cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or             (C₁₋₆alkyl)Het and heterocycle being optionally substituted             with R¹⁵⁰;         -   i) COR¹²⁷ wherein R¹²⁷ is H, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl             or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl             or (C₁₋₆alkyl)Het, said alkyl, cycloalkyl, aryl, Het,             (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het being optionally             substituted with R¹⁵⁰;         -   j) COOR¹²⁸ wherein R¹¹² is (C₁₋₆)alkyl substituted with             R¹⁵⁰, (C₃₋₇)cycloalkyl, or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl,             aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, said             (C₃₋₇)cycloalkyl, or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl,             Het, (C₁₋₆alkyl)aryl and (C₁₋₆alkyl)Het being optionally             substituted with R¹⁵⁰;         -   k) CONR¹²⁹R¹³⁰ wherein R¹²⁹ and R¹³⁰ are independently H,             (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl,             aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, or both R¹²⁹             and R¹³⁰ are covalently bonded together and to the nitrogen             to which they are attached to form a 5, 6 or 7-membered             saturated heterocycle, said alkyl, cycloalkyl,             alkyl-cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl, (C₁₋₆alkyl)Het             and heterocycle being optionally substituted with R¹⁵⁰;         -   l) aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, all of             which being optionally substituted with R¹⁵⁰;     -   1 to 3 substituents selected from: halogen, NO₂, cyano, azido or     -   1 to 3 substituents selected from:         -   a) (C₁₋₆)alkyl or haloalkyl, (C₃₋₇)cycloalkyl, C₃₋₇             spirocycloalkyl optionally containing 1 or 2 heteroatom,             (C₂₋₆)alkenyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, all of which             optionally substituted with R¹⁶⁰;         -   b) OR¹⁰⁴ wherein R¹⁰⁴ is H, (C₁₋₆alkyl), (C₃₋₇)cycloalkyl,             or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl             or (C₁₋₆alkyl)Het, said alkyl, cycloalkyl, aryl, Het,             (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het being optionally             substituted with R¹⁶⁰;         -   d) SO₃H, SO₂N(R¹⁰⁸)₂ or SO₂N(R¹⁰⁸)C(O)R¹⁰⁸ wherein each R¹⁰⁸             is independently H, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl or             (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or             (C₁₋₆alkyl)Het or both R¹⁰⁸ are covalently bonded together             and to the nitrogen to which they are attached to form a 5,             6 or 7-membered saturated heterocycle, said alkyl,             cycloalkyl, aryl, Het, (C₁₋₆)alkyl)aryl or (C₁₋₆alkyl)Het or             heterocycle being optionally substituted with R¹⁶⁰;         -   e) NR¹¹¹ R¹¹² wherein R¹¹¹ is H, (C₁₋₆)alkyl,             (C₃₋₇)cycloalkyl or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het,             (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, and R¹¹² is H,             (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl or             (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl,             (C₁₋₆alkyl)Het , COOR¹¹⁵ or SO₂R¹¹⁵ wherein R¹¹⁵ is             (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, or             (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or             (C₁₋₆alkyl)Het, or both R¹¹¹ and R¹¹² are covalently bonded             together and to the nitrogen to which they are attached to             form a 5, 6 or 7-membered saturated heterocycle, said alkyl,             cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, or             heterocycle being optionally substituted with R¹⁶⁰;         -   f) NR¹¹⁶COR¹¹⁷ wherein R¹¹⁶ and R¹¹⁷ is each H, (C₁₋₆)alkyl,             (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het,             (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, said (C₁₋₆)alkyl,             (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het,             (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het being optionally             substituted with R¹⁶⁰;         -   g) NR¹¹⁸CONR¹¹⁹R¹²⁰, wherein R¹¹⁸, R¹¹⁹ and R¹²⁰ is each H,             (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl,             aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, or R¹¹⁹ and             R¹²⁰ are covalently bonded together and to the nitrogen to             which they are attached to form a 5, 6 or 7-membered             saturated heterocycle; said alkyl, cycloalkyl,             (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or             (C₁₋₆alkyl)Het or heterocycle being optionally substituted             with R¹⁶⁰;         -   h) NR¹²¹COCOR¹²² wherein R¹²¹ is H, (C₁₋₆)alkyl optionally             substituted with R¹⁶⁰; or R¹²² is OR¹²³ or N(R¹²⁴)₂ wherein             R¹²³ and each R¹²⁴ is independently H, (C₁₋₆alkyl),             (C₃₋₇)cycloalkyl, or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl,             Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, or R¹²⁴ is OH or             O(C₁₋₆alkyl) or both R¹²⁴ are covalently bonded together to             form a 5, 6 or 7-membered saturated heterocycle, said alkyl,             cycloalkyl, alkyl-cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or             (C₁₋₆alkyl)Het and heterocycle being optionally substituted             with R¹⁶⁰;         -   j) tetrazole, COOR¹²⁸ wherein R¹²⁸ is H, (C₁₋₆)alkyl,             (C₃₋₇)cycloalkyl, or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl,             Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, said (C₁₋₆)alkyl,             (C₃₋₇)cycloalkyl, or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl,             Het, (C₁₋₆alkyl)aryl and (C₁₋₆alkyl)Het being optionally             substituted with R¹⁶⁰; and         -   k) CONR¹²⁹R¹³⁰ wherein R¹²⁹ and R¹³⁰ are independently H,             (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl,             aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, or both R¹²⁹             and R¹³⁰ are covalently bonded together and to the nitrogen             to which they are attached to form a 5, 6 or 7-membered             saturated heterocycle, said alkyl, cycloalkyl,             alkyl-cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl, (C₁₋₆alkyl)Het             and heterocycle being optionally substituted with R¹⁶⁰;             -   wherein R¹⁶⁰ is defined as 1 or 2 substituents selected                 from: tetrazole, halogen, CN, C₁₋₆alkyl, haloalkyl,                 COOR¹⁶¹, SO₃H, SO₂R¹⁶¹, OR¹⁶¹, N(R¹⁶²)₂, SO₂N(R¹⁶²)₂, or                 CON(R¹⁶²)₂, wherein R¹⁶¹ and each R¹⁶² is independently                 H, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl or                 (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl; or both R¹⁶² are                 covalently bonded together and to the nitrogen to which                 they are attached to form a 5, 6 or 7-membered saturated                 heterocycle; -   or Z is N(R^(6a))R⁶, wherein R^(6a) is H or (C₁₋₆alkyl); and -   R⁶ is (C₁₋₆)alkyl optionally substituted with:     -   1 to 4 substituents selected from: OPO₃H, NO₂, cyano, azido,         C(═NH)NH₂, C(═NH)NH(C₁₋₆)alkyl or C(═NH)NHCO(C₁₋₆)alkyl; or     -   1 to 4 substituents selected from:         -   a) (C₁₋₆)alkyl substituted with R^(150a), haloalkyl,             (C₃₋₇)cycloalkyl, C₃₋₇ spirocycloalkyl optionally containing             1 or 2 heteroatom, (C₂₋₆)alkenyl, (C₂₋₈)alkynyl,             (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, all of which optionally             substituted with R¹⁵⁰, wherein R^(150a) is the same as R¹⁵⁰             but is not halogen, OR^(150b), COOR^(150b), N(R^(150b))₂,             wherein R^(150b) is H or C₁₋₆alkyl;         -   b) OR¹⁰⁴ wherein R¹⁰⁴ is (C₁₋₆alkyl) substituted with R¹⁵⁰,             (C₃₋₇)cycloalkyl, or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl,             Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, said cycloalkyl,             aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het being             optionally substituted with R¹⁵⁰;         -   c) OCOR¹⁰⁵ wherein R¹⁰⁵ is (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl,             (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, Het, (C₁₋₆alkyl)aryl or             (C₁₋₆alkyl)Het, said alkyl, cycloalkyl, aryl, Het,             (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het being optionally             substituted with R¹⁵⁰;         -   d) SO₃H, SO₂N(R¹⁰⁸)₂ or SO₂N(R¹⁰⁸)C(O)R¹⁰⁸ wherein each R¹⁰⁸             is independently H, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl or             (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or             (C₁₋₆alkyl)Het or both R¹⁰⁸ are covalently bonded together             and to the nitrogen to which they are attached to form a 5,             6 or 7-membered saturated heterocycle, said alkyl,             cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het or             heterocycle being optionally substituted with R¹⁵⁰;         -   e) NR¹¹¹ R¹¹² wherein R¹¹¹ is (C₁₋₆)alkyl substituted with             R¹⁵⁰, (C₃₋₇)cycloalkyl or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl,             aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, and R¹¹² is H,             CN, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl or             (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl,             (C₁₋₆alkyl)Het or R¹¹¹ is H and R¹¹² is SO₂R¹¹⁵wherein R¹¹¹             is (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, or             (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or             (C₁₋₆alkyl)Het, or both R¹¹¹ and R¹¹² are covalently bonded             together and to the nitrogen to which they are attached to             form a 5, 6 or 7-membered saturated heterocycle, said             cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, or             heterocycle being optionally substituted with R¹⁵⁰;         -   f) NR¹¹⁶COR¹¹⁷ wherein R¹¹⁶ and R¹¹⁷ is each H, (C₁₋₆)alkyl,             (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het,             (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, said (C₁₋₆)alkyl,             (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het,             (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het being optionally             substituted with R¹⁵⁰;         -   g) NR¹¹⁸CONR¹¹⁹R¹²⁰, wherein R¹¹⁸, R¹¹⁹ and R¹²⁰ is each H,             (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl,             aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, or R¹¹⁸ is             covalently bonded to R¹¹⁹ and to the nitrogen to which they             are attached to form a 5, 6 or 7-membered saturated             heterocycle; or R¹¹¹ and R¹²⁰ are covalently bonded together             and to the nitrogen to which they are attached to form a 5,             6 or 7-membered saturated heterocycle; said alkyl,             cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het,             (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het or heterocycle being             optionally substituted with R¹⁵⁰;         -   h) NR¹²¹COCOR¹²² wherein R¹²¹ and R¹²² is each is H,             (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl,             a 6- or 10-membered aryl, Het, (C₁₋₆)alkyl)aryl or             (C₁₋₆alkyl)Het, said alkyl, cycloalkyl, alkyl-cycloalkyl,             aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het being             optionally substituted with R¹⁵⁰; or R¹²² is OR¹²³ or             N(R¹²⁴)₂ wherein R¹²³ and each R¹²⁴ is independently H,             (C₁₋₆alkyl), (C₃₋₇)cycloalkyl, or             (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or             (C₁₋₆alkyl)Het, or R¹²⁴ is OH or O(C₁₋₆alkyl) or both R¹²⁴             are covalently bonded together to form a 5, 6 or 7-membered             saturated heterocycle, said alkyl, cycloalkyl,             alkyl-cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or             (C₁₋₆alkyl)Het and heterocycle being optionally substituted             with R¹⁵⁰;         -   i) COR¹²⁷ wherein R¹²⁷ is H, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl             or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl             or (C₁₋₆alkyl)Het, said alkyl, cycloalkyl, aryl, Het,             (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het being optionally             substituted with R¹⁵⁰;         -   j) COOR¹²⁸ wherein R¹²⁸ is (C₁₋₆)alkyl substituted with             R¹⁵⁰, (C₃₋₇)cycloalkyl, or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl,             aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, said             (C₃₋₇)cycloalkyl, or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl,             Het, (C₁₋₆alkyl)aryl and (C₁₋₆alkyl)Het being optionally             substituted with R¹⁵⁰;         -   k) CONR¹²⁹R¹³⁰ wherein R¹²⁹ and R¹³⁰ are independently H,             (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl,             aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, or both R¹²⁹             and R¹³⁰ are covalently bonded together and to the nitrogen             to which they are attached to form a 5, 6 or 7-membered             saturated heterocycle, said alkyl, cycloalkyl,             alkyl-cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl, (C₁₋₆alkyl)Het             and heterocycle being optionally substituted with R¹⁵⁰;         -   l) aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, all of             which being optionally substituted with R¹⁵⁰; and     -   wherein R¹⁵⁰ is selected from:     -   1 to 3 substituents selected from: halogen, NO₂, cyano, azido or     -   1 to 3 substituents selected from:         -   a) (C₁₋₆)alkyl or haloalkyl, (C₃₋₇)cycloalkyl, C₃₋₁             spirocycloalkyl optionally containing 1 or 2 heteroatom,             (C₂₋₆)alkenyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, all of which             optionally substituted with R¹⁶⁰;         -   b) OR¹⁰⁴ wherein R¹⁰⁴ is H, (C₁₋₆alkyl), (C₃₋₇)cycloalkyl,             or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl             or (C₁₋₆alkyl)Het, said alkyl, cycloalkyl, aryl, Het,             (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het being optionally             substituted with R¹⁶⁰;         -   d) SO₃H, SO₂N(R¹⁰⁸)₂ or SO₂N(R¹⁰⁸)C(O)R¹⁰⁸ wherein each R¹⁰⁸             is independently H, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl or             (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆-alkyl)aryl or             (C₁₋₆alkyl)Het or both R¹⁰⁸ are covalently bonded together             and to the nitrogen to which they are attached to form a 5,             6 or 7-membered saturated heterocycle, said alkyl,             cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het or             heterocycle being optionally substituted with R¹⁶⁰;         -   e) NR¹¹¹R¹¹² wherein R¹¹¹ is H, (C₁₋₆)alkyl,             (C₃₋₇)cycloalkyl or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het,             (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, and R¹¹² is H,             (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl or             (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl,             (C₁₋₆alkyl)Het, COOR¹¹⁵ or SO₂R¹¹⁵ wherein R¹¹⁵ is             (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, or             (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or             (C₁₋₆alkyl)Het, or both R¹¹¹ and R¹¹² are covalently bonded             together and to the nitrogen to which they are attached to             form a 5, 6 or 7-membered saturated heterocycle, said alkyl,             cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, or             heterocycle being optionally substituted with R¹⁶⁰;         -   f) NR¹¹⁶COR¹¹⁷ wherein R¹¹⁶ and R¹¹⁷ is each H, (C₁₋₆)alkyl,             (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het,             (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, said (C₁₋₆)alkyl,             (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het,             (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het being optionally             substituted with R¹⁶⁰;         -   g) NR¹¹⁸CONR¹¹⁹R¹²⁰, wherein R¹¹⁸, R¹¹⁹ and R¹²⁰ is each H,             (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl,             aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆-alkyl)Het, or R¹¹⁹ and             R¹²⁰ are covalently bonded together and to the nitrogen to             which they are attached to form a 5, 6 or 7-membered             saturated heterocycle; said alkyl, cycloalkyl,             (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or             (C₁₋₆alkyl)Het or heterocycle being optionally substituted             with R¹⁶⁰;         -   h) NR¹²¹COCOR¹²² wherein R¹²¹ is H, (C₁₋₆)alkyl optionally             substituted with R¹⁶⁰; or R¹²² is OR¹²³ or N(R¹²⁴)₂ wherein             R¹²³ and each R¹²⁴ is independently H, (C₁₋₆alkyl),             (C₃₋₇)cycloalkyl, or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl,             Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, or R¹²⁴ is OH or             O(C₁₋₆alkyl) or both R¹²⁴ are covalently bonded together to             form a 5, 6 or 7-membered saturated heterocycle, said alkyl,             cycloalkyl, alkyl-cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or             (C₁₋₆alkyl)Het and heterocycle being optionally substituted             with R¹⁶⁰;         -   j) tetrazole, COOR¹²⁸ wherein R¹²⁸ is H, (C₁₋₆)alkyl,             (C₃₋₇)cycloalkyl, or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl,             Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, said (C₁₋₆)alkyl,             (C₃₋₇)cycloalkyl, or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl,             Het, (C₁₋₆alkyl)aryl and (C₁₋₆alkyl)Het being optionally             substituted with R¹⁶⁰; and         -   k) CONR¹²⁹R¹³⁰ wherein R¹²⁹ and R¹³⁰ are independently H,             (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl,             aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, or both R¹²⁹             and R¹³⁰ are covalently bonded together and to the nitrogen             to which they are attached to form a 5, 6 or 7-membered             saturated heterocycle, said alkyl, cycloalkyl,             alkyl-cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl, (C₁₋₆alkyl)Het             and heterocycle being optionally substituted with R¹⁶⁰;             -   wherein R¹⁶⁰ is defined as 1 or 2 substituents selected                 from: tetrazole, halogen, CN, C₁₋₆alkyl, haloalkyl,                 COOR¹⁶¹, SO₃H, SO₂R¹⁶¹, OR¹⁶¹, N(R¹⁶²)₂, SO₂N(R¹⁶²)₂, or                 CON(R¹⁶²)₂, wherein R¹⁶¹ and each R¹⁶² is independently                 H, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl or                 (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl; or both R¹⁶² are                 covalently bonded together and to the nitrogen to which                 they are attached to form a 5, 6 or 7-membered saturated                 heterocycle. -   or R⁶ is

wherein, preferably, R⁷ and R⁸ are each independently H, (C₁₋₆)alkyl, haloalkyl, (C₃₋₇)cycloalkyl, 6- or 10-membered aryl, Het, (C₁₋₆)alkyl-aryl, (C₁₋₆)alkyl-Het, wherein said alkyl, cycloalkyl, aryl, Het, (C₁₋₆)alkyl-aryl, (C₁₋₆)alkyl-Het are optionally substituted with R⁷⁰; or

-   R⁷ and R⁸ are covalently bonded together to form second     (C₃₋₇)cycloalkyl or a 4, 5- or 6-membered heterocycle having from 1     to 3 heteroatom selected from O, N, and S; or when Z is N(R^(6a))R⁶,     either of R⁷ or R⁸ is covalently bonded to R^(6a) to form a     nitrogen-containing 5- or 6-membered heterocycle;     -   wherein, preferably, R⁷⁰ is selected from:     -   1 to 4 substituents selected from: halogen, NO₂, cyano, azido;         or     -   1 to 4 substituents selected from:         -   a) (C₁₋₆)alkyl or haloalkyl, (C₃₋₇)cycloalkyl, C₃₋₇             spirocycloalkyl optionally containing 1 or 2 heteroatom,             (C₂₋₆)alkenyl, (C₂₋₈)alkynyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl,             all of which optionally substituted with R¹⁵⁰;         -   b) OR¹⁰⁴ wherein R¹⁰⁴ is H, (C₁₋₆alkyl), (C₃₋₇)cycloalkyl,             or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl             or (C₁₋₆alkyl)Het, said alkyl, cycloalkyl, aryl, Het,             (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het being optionally             substituted with R¹⁵⁰;         -   d) SO₂N(R¹⁰⁸)₂ or SO₂N(R¹⁰⁸)C(O)R¹⁰⁸ wherein each R¹⁰⁸ is             independently H, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl or             (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or             (C₁₋₆alkyl)Het or both R¹⁰⁸ are covalently bonded together             and to the nitrogen to which they are attached to form a 5,             6 or 7-membered saturated heterocycle, said alkyl,             cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het or             heterocycle being optionally substituted with R¹⁵⁰;         -   e) NR¹¹¹ R¹¹² wherein R¹¹¹ is H, (C₁₋₆)alkyl,             (C₃₋₇)cycloalkyl or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het,             (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, and R¹¹² is H, CN,             (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl or             (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl,             (C₁₋₆alkyl)Het, COOR¹¹⁵ or SO₂R¹¹⁵wherein R¹¹⁵ is             (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, or             (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or             (C₁₋₆alkyl)Het, or both R¹¹¹ and R¹¹² are covalently bonded             together and to the nitrogen to which they are attached to             form a 5, 6 or 7-membered saturated heterocycle, said alkyl,             cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, or             heterocycle being optionally substituted with R¹⁵⁰;         -   f) NR¹¹⁶COR¹¹⁷ wherein R¹¹⁶ and R¹¹⁷ is each H, (C₁₋₆)alkyl,             (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het,             (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, said (C₁₋₆)alkyl,             (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het,             (C₁₋₆alkyl)aryl or (C₁₋₆-alkyl)Het being optionally             substituted with R¹⁵⁰;         -   g) NR¹¹⁸CONR¹¹⁹R¹²⁰, wherein R¹¹⁸, R¹¹⁹ and R¹²⁰ is each H,             (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl,             aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, or R¹¹⁸ is             covalently bonded to R¹¹⁹ and to the nitrogen to which they             are attached to form a 5, 6 or 7-membered saturated             heterocycle; or R¹¹⁹ and R¹²⁰ are covalently bonded together             and to the nitrogen to which they are attached to form a 5,             6 or 7-membered saturated heterocycle; said alkyl,             cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het,             (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het or heterocycle being             optionally substituted with R¹⁵⁰;         -   h) NR¹²¹COCOR¹²² wherein R¹²¹ is H, (C₁₋₆)alkyl,             (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, a 6- or             10-membered aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het,             said alkyl, cycloalkyl, alkyl-cycloalkyl, aryl, Het,             (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het being optionally             substituted with R¹⁵⁰; and R¹²² is OR¹²³ or N(R¹²⁴)₂ wherein             R¹²³ and each R¹²⁴ is independently H, (C₁₋₆alkyl),             (C₃₋₇)cycloalkyl, or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl,             Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, or R¹²⁴ is OH or             O(C₁₋₆alkyl) or both R¹²⁴ are covalently bonded together to             form a 5, 6 or 7-membered saturated heterocycle, said alkyl,             cycloalkyl, alkyl-cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or             (C₁₋₆alkyl)Het and heterocycle being optionally substituted             with R¹⁵⁰;         -   i) COR¹²⁷ wherein R¹²⁷ is H, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl             or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl             or (C₁₋₆alkyl)Het, said alkyl, cycloalkyl, aryl, Het,             (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het being optionally             substituted with R¹⁵⁰;         -   j) COOR¹²⁸ wherein R¹²⁸ is H, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl,             or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl             or (C₁₋₆alkyl)Het, said (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, or             (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl and             (C₁₋₆alkyl)Het being optionally substituted with R¹⁵⁰;         -   k) CONR¹²⁹R¹³⁰ wherein R¹²⁹ and R¹³⁰ are independently H,             (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl,             aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, or both R¹²⁹             and R¹³⁰ are covalently bonded together and to the nitrogen             to which they are attached to form a 5, 6 or 7-membered             saturated heterocycle, said alkyl, cycloalkyl,             alkyl-cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl, (C₁₋₆alkyl)Het             and heterocycle being optionally substituted with R¹⁵⁰;         -   l) aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, all of             which being optionally substituted with R¹⁵⁰;     -   wherein, preferably, R¹⁵⁰ is selected from:     -   1 to 3 substituents selected from: halogen, NO₂, cyano, azido;         or     -   1 to 3 substituents selected from:         -   a) (C₁₋₆)alkyl or haloalkyl, (C₃₋₇)cycloalkyl, C₃₋₇             spirocycloalkyl optionally containing 1 or 2 heteroatom,             (C₂₋₆)alkenyl, (C₂₋₈)alkynyl, all of which optionally             substituted with R¹⁶⁰;         -   b) OR¹⁰⁴ wherein R¹⁰⁴ is H, (C₁₋₆alkyl) or (C₃₋₇)cycloalkyl,             said alkyl and cycloalkyl being optionally substituted with             R¹⁶⁰;         -   d) SO₂N(R¹⁰⁸)₂ wherein R¹⁰⁸ is H, (C₁₋₆)alkyl or             (C₃₋₇)cycloalkyl, said alkyl or cycloalkyl being optionally             substituted with R¹⁶⁰;         -   e) NR¹¹¹ R¹¹² wherein R¹¹¹ is H, (C₁₋₆)alkyl or             (C₃₋₇)cycloalkyl, and R¹¹² is H, (C₁₋₆)alkyl,             (C₃₋₇)cycloalkyl or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het,             (C₁₋₆alkyl)aryl, (C₁₋₆alkyl)Het, COOR¹¹⁵ or SO₂R¹¹⁵ wherein             R¹¹⁵ is (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, or             (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or             (C₁₋₆alkyl)Het, or both R¹¹¹ and R¹¹² are covalently bonded             together and to the nitrogen to which they are attached to             form a 5, 6 or 7-membered saturated heterocycle, said alkyl,             cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, or             heterocycle being optionally substituted with R¹⁶⁰;         -   f) NR¹¹⁶COR¹¹⁷ wherein R¹¹⁶ and R¹¹⁷ is each H, (C₁₋₆)alkyl             or (C₃₋₇)cycloalkyl, said (C₁₋₆)alkyl or (C₃₋₇)cycloalkyl             being optionally substituted with R¹⁶⁰;         -   g) NR¹¹⁸CONR¹¹⁹R¹²⁰, wherein R¹¹⁸, R¹¹⁹ and R¹²⁰ is each H,             (C₁₋₆)alkyl or (C₃₋₇)cycloalkyl; or R¹¹⁹ and R¹²⁰ are             covalently bonded together and to the nitrogen to which they             are attached to form a 5, 6 or 7-membered saturated             heterocycle; said alkyl, cycloalkyl or heterocycle being             optionally substituted with R¹⁶⁰;         -   h) NR¹²¹COCOR¹²² wherein R¹²¹ is H, (C₁₋₆)alkyl or             (C₃₋₇)cycloalkyl, said alkyl or cycloalkyl being optionally             substituted with R¹⁶⁰; or R¹²² is OR¹²³ or N(R¹²⁴)₂ wherein             R¹²³ and each R¹²⁴ is independently H, (C₁₋₆alkyl) or             (C₃₋₇)cycloalkyl, or R¹²⁴ is OH or O(C₁₋₆alkyl) or both R¹²⁴             are covalently bonded together to form a 5, 6 or 7-membered             saturated heterocycle, said alkyl, cycloalkyl and             heterocycle being optionally substituted with R¹⁶⁰;         -   j) tetrazole, COOR¹²⁸ wherein R¹²⁸ is H, (C₁₋₆)alkyl or             (C₃₋₇)cycloalkyl, said (C₁₋₆)alkyl and (C₃₋₇)cycloalkyl             being optionally substituted with R¹⁶⁰; and         -   k) CONR¹²⁹R¹³⁰ wherein R¹²⁹ and R¹³⁰ are independently H,             (C₁₋₆)alkyl or (C₃₋₇)cycloalkyl, or both R¹²⁹ and R¹³⁰ are             covalently bonded together and to the nitrogen to which they             are attached to form a 5, 6 or 7-membered saturated             heterocycle, said alkyl, cycloalkyl and heterocycle being             optionally substituted with R¹⁶⁰;             -   wherein R¹⁶⁰ is defined as 1 or 2 substituents selected                 from: tetrazole, halogen, CN, C₁₋₆alkyl, haloalkyl,                 COOR¹⁶¹, OR¹⁶¹, N(R¹⁶²)₂ or CON(R¹⁶²)₂, wherein R¹⁶¹ and                 each R¹⁶² is independently H or (C₁₋₆)alkyl; -   R⁹ is H; or R⁹ is covalently bonded to either of R⁷ or R⁸ to form a     5- or 6-membered heterocycle; and -   Q is a 6- or 10-membered aryl, Het, all of which being optionally     substituted with:

wherein R¹⁰⁰ is:

-   -   1 to 4 substituents selected from: halogen, NO₂, cyano or azido;         or     -   1 to 4 substituents selected from:         -   a) (C₁₋₆)alkyl or haloalkyl, (C₃₋₇)cycloalkyl,             (C₂₋₆)alkenyl, (C₂₋₈)alkynyl, (C₁₋₆) alkyl-(C₃₋₇)cycloalkyl,             all of which optionally substituted with R¹⁵⁰;         -   b) OR¹⁰⁴ wherein R¹⁰⁴ is H, (C₁₋₆alkyl), (C₃₋₇)cycloalkyl,             or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl             or (C₁₋₆alkyl)Het, said alkyl, cycloalkyl, aryl, Het,             (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het being optionally             substituted with R¹⁵⁰;         -   e) NR¹¹¹R¹¹² wherein R¹¹¹ is H, (C₁₋₆)alkyl,             (C₃₋₇)cycloalkyl or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het,             (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, and R¹¹² is H, CN,             (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl or             (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl,             (C₁₋₆alkyl)Het, COOR¹¹⁵ or SO₂R¹¹⁵wherein R¹¹⁵ is             (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, or             (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or             (C₁₋₆alkyl)Het, or both R¹¹¹ and R¹¹² are covalently bonded             together and to the nitrogen to which they are attached to             form a 5, 6 or 7-membered saturated heterocycle, said alkyl,             cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, or             heterocycle being optionally substituted with R¹⁵⁰;         -   f) NR¹¹⁶COR¹¹⁷ wherein R¹¹⁶ and R¹¹⁷ is each H, (C₁₋₆)alkyl,             (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het,             (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, said (C₁₋₆)alkyl,             (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het,             (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het being optionally             substituted with R¹⁵⁰;         -   g) NR¹¹⁸CONR¹¹⁹R¹²⁰, wherein R¹¹⁸, R¹¹⁹ and R¹²⁰ is each H,             (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl,             aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, or R¹¹⁸ is             covalently bonded to R¹¹⁹ and to the nitrogen to which they             are attached to form a 5, 6 or 7-membered saturated             heterocycle; or R¹¹⁹ and R¹²⁰ are covalently bonded together             and to the nitrogen to which they are attached to form a 5,             6 or 7-membered saturated heterocycle; said alkyl,             cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het,             (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het or heterocycle being             optionally substituted with R¹⁵⁰;         -   h) NR¹²¹COCOR¹²² wherein R¹²¹ and R¹²² is each is H,             (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl,             a 6- or 10-membered aryl, Het, (C₁₋₆alkyl)aryl or             (C₁₋₆alkyl)Het, said alkyl, cycloalkyl, alkyl-cycloalkyl,             aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het being             optionally substituted with R¹⁵⁰; or R¹²² is OR¹²³ or             N(R¹²⁴)₂ wherein R¹²³ and each R¹²⁴ is independently H,             (C₁₋₆alkyl), (C₃₋₇)cycloalkyl, or             (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or             (C₁₋₆alkyl)Het, or R¹²⁴ is OH or O(C₁₋₆alkyl) or both R¹²⁴             are covalently bonded together to form a 5, 6 or 7-membered             saturated heterocycle, said alkyl, cycloalkyl,             alkyl-cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or             (C₁₋₆alkyl)Het and heterocycle being optionally substituted             with R¹⁵⁰;         -   j) COOR¹²⁸ wherein R¹²⁸ is H, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl,             or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl             or (C₁₋₆alkyl)Het, said (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, or             (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl and             (C₁₋₆alkyl)Het being optionally substituted with R¹⁵⁰;         -   k) CONR¹²⁹R¹³⁰ wherein R¹²⁹ and R¹³⁰ are independently H,             (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl,             aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, or both R¹²⁹             and R¹³⁰ are covalently bonded together and to the nitrogen             to which they are attached to form a 5, 6 or 7-membered             saturated heterocycle, said alkyl, cycloalkyl,             alkyl-cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl, (C₁₋₆alkyl)Het             and heterocycle being optionally substituted with R¹⁵⁰;         -   l) aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, all of             which being optionally substituted with R¹⁵⁰;     -   wherein R¹⁵⁰ is selected from:     -   1 to 3 substituents selected from: halogen, NO₂, cyano or azido;         or     -   1 to 3 substituents selected from:         -   a) (C₁₋₆)alkyl or haloalkyl, (C₃₋₇)cycloalkyl, C₃₋₇             spirocycloalkyl optionally containing 1 or 2 heteroatom,             (C₂₋₆)alkenyl, (C₂₋₈)alkynyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl,             all of which optionally substituted with R¹⁶⁰;         -   b) OR¹⁰⁴ wherein R¹⁰⁴ is H, (C₁₋₆alkyl), (C₃₋₇)cycloalkyl,             or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl             or (C₁₋₆alkyl)Het, said alkyl, cycloalkyl, aryl, Het,             (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het being optionally             substituted with R¹⁶⁰;         -   d) SO₃H, SO₂N(R¹⁰⁸)₂ or SO₂N(R¹⁰⁸)C(O)R¹⁰⁸ wherein each R¹⁰⁸             is independently H, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl or             (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or             (C₁₋₆alkyl)Het or both R¹⁰⁸ are covalently bonded together             and to the nitrogen to which they are attached to form a 5,             6 or 7-membered saturated heterocycle, said alkyl,             cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het or             heterocycle being optionally substituted with R¹⁶⁰;         -   e) NR¹¹¹R¹¹² wherein R¹¹¹ is H, (C₁₋₆)alkyl,             (C₃₋₇)cycloalkyl or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het,             (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, and R¹¹² is H, CN,             (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl or             (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl,             (C₁₋₆alkyl)Het or SO₂R¹¹⁵ wherein R¹¹⁵ is (C₁₋₆)alkyl,             (C₃₋₇)cycloalkyl, or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl,             Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, or both R¹¹¹ and             R¹¹² are covalently bonded together and to the nitrogen to             which they are attached to form a 5, 6 or 7-membered             saturated heterocycle, said alkyl, cycloalkyl, aryl, Het,             (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, or heterocycle being             optionally substituted with R¹⁶⁰;         -   f) NR¹¹⁶COR¹¹⁷ wherein R¹¹⁶ and R¹¹⁷ is each H, (C₁₋₆)alkyl,             (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het,             (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, said (C₁₋₆)alkyl,             (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het,             (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het being optionally             substituted with R¹⁶⁰;         -   g) NR¹¹⁸CONR¹¹⁹R¹²⁰, wherein R¹¹⁸, R¹¹⁹ and R¹²⁰ is each H,             (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl,             aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, or R¹¹⁸ is             covalently bonded to R¹¹⁹ and to the nitrogen to which they             are attached to form a 5, 6 or 7-membered saturated             heterocycle; or R¹¹⁹ and R¹²⁰ are covalently bonded together             and to the nitrogen to which they are attached to form a 5,             6 or 7-membered saturated heterocycle; said alkyl,             cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het,             (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het or heterocycle being             optionally substituted with R¹⁶⁰;         -   h) NR¹²¹COCOR¹²² wherein R¹²¹ is H, (C₁₋₆)alkyl optionally             substituted with R¹⁶⁰; or R¹²² is OR¹²³ or N(R¹²⁴)₂ wherein             R¹²³ and each R¹²⁴ is independently H, (C₁₋₆alkyl),             (C₃₋₇)cycloalkyl, or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl,             Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, or R¹²⁴ is OH or             O(C₁₋₆alkyl) or both R¹²⁴ are covalently bonded together to             form a 5, 6 or 7-membered saturated heterocycle, said alkyl,             cycloalkyl, alkyl-cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or             (C₁₋₆alkyl)Het and heterocycle being optionally substituted             with R¹⁶⁰;         -   j) tetrazole, COOR¹²⁸ wherein R¹²⁸ is H, (C₁₋₆)alkyl,             (C₃₋₇)cycloalkyl, or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl,             Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, said (C₁₋₆)alkyl,             (C₃₋₇)cycloalkyl, or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl,             Het, (C₁₋₆alkyl)aryl and (C₁₋₆alkyl)Het being optionally             substituted with R¹⁶⁰; and         -   k) CONR¹²⁹R¹³⁰ wherein R¹²⁹ and R¹³⁰ are independently H,             (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl,             aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, or both R¹²⁹             and R¹³⁰ are covalently bonded together and to the nitrogen             to which they are attached to form a 5, 6 or 7-membered             saturated heterocycle, said alkyl, cycloalkyl,             alkyl-cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl, (C₁₋₆alkyl)Het             and heterocycle being optionally substituted with R¹⁶⁰;             -   wherein R¹⁶⁰ is defined as 1 or 2 substituents selected                 from: tetrazole, halogen, CN, C₁₋₆alkyl, haloalkyl,                 COOR¹⁶¹, SO₃H, SR¹⁶¹, SO₂R¹⁶¹, OR¹⁶¹, N(R¹⁶²)₂,                 SO₂N(R¹⁶²)₂, or CON(R¹⁶²)₂, wherein R¹⁶¹ and each R¹⁶²                 is independently H, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl or                 (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl; or both R¹⁶² are                 covalently bonded together and to the nitrogen to which                 they are attached to form a 5, 6 or 7-membered saturated                 heterocycle;                 or a salt thereof;                 wherein said probe comprises a detectable label attached                 to any suitable position, whereby said probe binds to an                 HCV polymerase or an analog thereof and is capable of                 being displaced by an inhibitor thereof.

More preferably, the probe of the invention is a compound of formula:

-   wherein R¹ is (C₅₋₆)cycloalkyl; -   R² is phenyl, or Het both being optionally substituted with R²⁰; -   R³, R⁷, R⁸, R⁹, R¹⁰⁰, and R¹⁵⁰ are as defined above; -   R¹¹ is OPO₃H, NO₂, cyano, azido, C(═NH)NH₂, C(═NH)NH(C₁₋₆)alkyl or     C(═NH)NHCO(C₁₋₆)alkyl; or     -   a) (C₁₋₆)alkyl substituted with R^(150a), haloalkyl,         (C₃₋₇)cycloalkyl, C₃₋₇ spirocycloalkyl optionally containing 1         or 2 heteroatom, (C₂₋₆)alkenyl, (C₂₋₈)alkynyl,         (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, all of which optionally         substituted with R¹⁵⁰, wherein R^(150a) is the same as R¹⁵⁰ but         is not halogen, OR^(150b), COOR^(150b), N(R^(150b))₂, wherein         R^(151b) is H or C₁₋₆alkyl;     -   b) OR¹⁰⁴ wherein R¹⁰⁴ is (C₁₋₆alkyl) substituted with R¹⁵⁰,         (C₃₋₇)cycloalkyl, or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het,         (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, said cycloalkyl, aryl, Het,         (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het being optionally substituted         with R¹⁵⁰;     -   c) OCOR¹⁰⁵ wherein R¹⁰⁵ is (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl,         (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, Het, (C₁₋₆alkyl)aryl or         (C₁₋₆alkyl)Het, said alkyl, cycloalkyl, aryl, Het,         (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het being optionally substituted         with R¹⁵⁰;     -   d) SO₃H, SO₂N(R¹⁰⁸)₂ or SO₂N(R¹⁰⁸)C(O)R¹⁰⁸ wherein each R¹⁰⁸ is         independently H, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl or         (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or         (C₁₋₆alkyl)Het or both R¹⁰⁶ are covalently bonded together and         to the nitrogen to which they are attached to form a 5, 6 or         7-membered saturated heterocycle, said alkyl, cycloalkyl, aryl,         Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het or heterocycle being         optionally substituted with R¹⁵⁰;     -   e) NR¹¹¹R¹¹² wherein R¹¹¹ is (C₁₋₆)alkyl substituted with R¹⁵⁰,         (C₃₋₇)cycloalkyl or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het,         (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, and R¹¹² is H, CN,         (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl,         aryl, Het, (C₁₋₆alkyl)aryl, (C₁₋₆alkyl)Het or     -   R¹¹¹ is H and R¹¹² is SO₂R¹¹⁵wherein R¹¹¹ is (C₁₋₆)alkyl,         (C₃₋₇)cycloalkyl, or (C₁₋₆)alkyl-(C₁₋₆)cycloalkyl, aryl, Het,         (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, or both R¹¹¹ and R¹¹² are         covalently bonded together and to the nitrogen to which they are         attached to form a 5, 6 or 7-membered saturated heterocycle,         said cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het,         or heterocycle being optionally substituted with R¹⁵⁰;     -   f) NR¹¹⁶COR¹¹⁷ wherein R¹¹⁶ and R¹¹⁷ is each H, (C₁₋₆)alkyl,         (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het,         (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, said (C₁₋₆)alkyl,         (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het,         (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het being optionally substituted         with R¹⁵⁰;     -   g) NR¹¹⁸CONR¹¹⁹R¹²⁰, wherein R¹¹⁸, R¹¹⁹ and R¹²⁰ is each H,         (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl,         aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, or R¹¹⁸ is         covalently bonded to R¹¹⁹ and to the nitrogen to which they are         attached to form a 5, 6 or 7-membered saturated heterocycle; or         R¹¹⁹ and R¹²⁰ are covalently bonded together and to the nitrogen         to which they are attached to form a 5, 6 or 7-membered         saturated heterocycle; said alkyl, cycloalkyl,         (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or         (C₁₋₆alkyl)Het or heterocycle being optionally substituted with         R¹⁵⁰;     -   h) NR¹²¹COCOR¹²² wherein R¹²¹ and R¹²² is each is H,         (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₄)cycloalkyl, a         6- or 10-membered aryl, Het, (C₁₋₆)alkyl)aryl or (C₁₋₆alkyl)Het,         said alkyl, cycloalkyl, alkyl-cycloalkyl, aryl, Het,         (C₁₋₆alkyl)aryl or (C₁₋₆-alkyl)Het being optionally substituted         with R¹⁵⁰; or R¹²² is OR¹²³ or N(R¹²⁴)₂ wherein R¹²³ and each         R¹²⁴ is independently H, (C₁₋₆alkyl), (C₃₋₇)cycloalkyl, or         (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or         (C₁₋₆alkyl)Het, or R¹²⁴ is OH or O(C₁₋₆alkyl) or both R¹²⁴ are         covalently bonded together to form a 5, 6 or 7-membered         saturated heterocycle, said alkyl, cycloalkyl, alkyl-cycloalkyl,         aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het and heterocycle         being optionally substituted with R¹⁵⁰;     -   i) COR¹²⁷ wherein R¹²⁷ is H, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl or         (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or         (C₁₋₆alkyl)Het, said alkyl, cycloalkyl, aryl, Het,         (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het being optionally substituted         with R¹⁵⁰;     -   j) COOR¹²⁸ wherein R¹²⁸ is (C₁₋₆)alkyl substituted with R¹⁵⁰,         (C₃₋₇)cycloalkyl, or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het,         (C₁₋₆alkyl)aryl or (C₁₋₆-alkyl)Het, said (C₃₋₇)cycloalkyl, or         (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl and         (C₁₋₆alkyl)Het being optionally substituted with R¹⁵⁰;     -   k) CONR¹²⁹R¹³⁰ wherein R¹²⁹ and R¹³⁰ are independently H,         (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl,         aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, or both R¹²⁹ and         R¹³⁰ are covalently bonded together and to the nitrogen to which         they are attached to form a 5, 6 or 7-membered saturated         heterocycle, said alkyl, cycloalkyl, alkyl-cycloalkyl, aryl,         Het, (C₁₋₆alkyl)aryl, (C₁₋₆alkyl)Het and heterocycle being         optionally substituted with R¹⁵⁰;     -   l) aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, all of which         being optionally substituted with R¹⁵⁰ -   wherein R¹⁵⁰ is as defined herein;     or a salt thereof;     wherein said compound is either:     -   a) marked with a radioactive isotope at any suitable position;     -   b) linked to a detectable moiety by a suitable linker at any         suitable position, except R¹and R³; or     -   c) linked to an affinity tag at any suitable position, except R¹         and R³.

Even more preferably, the probe of the invention is a compound of formula:

wherein

-   R¹ is (C₅₋₆)cycloalkyl; -   R² is phenyl, or Het both being optionally substituted with R²⁰; -   R³ and R¹⁵⁰ are as defined above;     or a salt thereof;     wherein said compound is optionally:     -   a) marked with a radioactive isotope at any suitable position;     -   b) linked to a detectable moiety by a suitable linker at any         suitable position, except R¹and R³; or     -   c) linked to an affinity tag at any suitable position, except         R¹and R³.

Specifically, according to a first aspect of the invention, the probe of the present invention is selected from the group consisting of:

According to an alternative aspect of this first embodiment, there is provided a method for identifying compounds that inhibit HCV polymerase comprising the steps of:

-   -   a) contacting an HCV polymerase or an analog thereof with a         probe of formula I, as defined herein, so as to form a complex         having said probe bound to said polymerase;     -   b) measuring the signal from said complex to establish a base         line level;     -   c) incubating the product of step a) with a test compound; and     -   d) measuring the signal from said complex; and     -   e) comparing the signal from step d) with the signal from step         b);         whereby a modulation in said signal is an indication that said         test compound inhibits said polymerase.

Preferably, the method for identifying compounds capable of inhibiting HCV polymerase, comprises:

-   -   f) repeating steps (a) to (e), as defined above in a high         throughput screen.

Alternatively, there is provided a probe of formula I:

-   A is O, S, NR³, or CR³; -   B is NR¹ or CR¹; with the proviso that, when A is CR³, B is NR¹, and     when A is O or S, B is CR¹; -   ----- represents either a single or a double bond; -   R¹ is selected from the group consisting of: (C₄₋₇)cycloalkyl     optionally substituted with (C₁₋₆ alkyl); norbornane, 5-, 6- or     7-membered heterocycle having 1 to 4 heteroatoms selected from O, N,     and S, all of which optionally substituted with 1 to 4 substituent     selected from the group consisting of:     -   halo, OH and C₁₋₆ alkyl optionally substituted with hydroxy; -   R² is selected from the group consisting of: phenyl,     pyridine-N-oxide, 5- or 6-membered aromatic heterocycle having 1 to     4 heteroatoms selected from O, N, and S, and 9- or 10-membered     aromatic heterobicycle having 1 to 4 heteroatoms selected from O, N     and S;     -   said phenyl, pyridine-N-oxide, aromatic heterocycle and aromatic         heterobicycle being optionally substituted with from 1 to 4         substituents selected from the group consisting of: halogen,         C₁₋₆ haloalkyl, (C₁₋₆)alkyl, C₁₋₆ alkoxy, OH, amino optionally         mono- or di-substituted with C₁₋₆ alkyl; -   R³ is selected from the group consisting of: H, (C₁₋₆)alkyl, (C₁₋₆     alkyl)-(C₆₋₁₀aryl), (C₁₋₆ alkyl)-5- or 6-membered heterocycle having     1 to 4 heteroatoms selected from O, N, and S, and 5- or 6-membered     heterocycle having 1 to 4 heteroatoms selected from O, N and S,     -   wherein said aryl and said heterocycle are optionally         substituted with from 1 to 4 substituents selected from the         group consisting of: COOH, COO(C₁₋₆ alkyl), halogen, and (C₁₋₄         alkyl); -   M is N, CR^(4a), or COR^(4b), wherein R^(4a) is selected from the     group consisting of: H, halogen, and (C₁₋₆ alkyl); and R^(4b) is     selected from the group consisting of: H and (C₁₋₆ alkyl); -   K and L is each independently N or CR⁶, wherein R⁶ is H, halo, C₁₋₆     alkyl, OH, or C₁₋₆ alkoxy; -   R⁵ is —C(Y)-Z, wherein Y is O or S; and Z is NHR^(5a) or OR^(5a);     wherein: -   R^(5a) is selected from the group consisting of: H, (C₁₋₆)alkyl,     (C₂₋₆)alkenyl, (C₃₋₆)cycloalkyl optionally substituted with     C₁₋₆alkyl or C₂₋₆alkenyl, (C₆₋₁₀)aryl optionally substituted with     C₁₋₆alkyl or C₂₋₆alkenyl, N-{(C₁₋₆)alkyl}₂,     NHCOO(C₁₋₆)alkyl(C₆₋₁₀)aryl, NHCO(C₆₋₁₀)aryl, -5- or 6-atom     heterocycle, having 1 to 4 heteroatoms selected from O, N and S, and     -9- or 10-atom heterobicycle having 1 to 4 heteroatoms selected from     O, N and S;     -   wherein said alkyl, alkenyl, cycloalkyl, aryl, heterocycle or         heterobicycle are all optionally substituted with from 1 to 4         substituents selected from: OH, COOH, (C₁₋₆)alkyl,         (C₂₋₄)alkenyl, (C₁₋₆)alkyl-hydroxy, COO(C₁₋₆)alkyl, C₃₋₇         cycloalkyl, benzyloxy, halogen, (C₂₋₄)alkenyl-(C₁₋₆)alkyl-COOH,         coumarin, (C₁₋₆)alkyl-amino, NH(C₁₋₆ alkyl), C(halogen)₃,         —C(O)NH(C₁₋₄)alkyl, and —C(O)NH(C₆₋₁₀)aryl, 5- or 6-membered         heterocycle having 1 to 4 heteroatoms selected from O, N and S,         9- or 10-membered heterobicycle having 1 to 4 heteroatoms         selected from O, N and S, and 6- or 10-membered aryl;         -   wherein said alkyl, alkenyl, cycloalkyl, aryl, heterocycle             and heterobicycle are all optionally substituted with from 1             to 4 substituents selected from: halogen, OPO₃H,             sulfonamido, SO₃H, SO₂CH₃, —CONH₂, —COCH₃, (C₁₋₃)alkyl,             (C₂₋₄alkenyl)COOH, tetrazolyl, COOH, —CONH₂, triazolyl, OH,             NO₂, NH₂, —O(C₁₋₆ alkyl)COOH, hydantoin, benzoyleneurea,             (C₁₋₄)alkoxy, cyano, azido, —O—(C₁₋₆)alkyl COOH,             —O—(C₁₋₆)alkyl COO—(C₁₋₆)alkyl, NHCO—(C₁₋₆alkyl), —NHCOCOOH,             —NHCOCONHOH, —NHCOCONH₂—NHCOCONHCH₃, —NHCO(C₁₋₆)alkyl-COOH,             —NHCOCONH(C₁₋₆)alkyl-COOH, —NHCO(C₃₋₇)cycloalkyl-COOH,             —NHCONH(C₆₋₁₀)aryl-COOH, —NHCONH(C₆₋₁₀)aryl-COO(C₁₋₆)alkyl,             —NHCONH(C₁₋₆)alkyl-COOH, —NHCONH(C₁₋₆)alkyl-COO(C₁₋₆)alkyl,             —NHCONH(C₁₋₆)alkyl-(C₂₋₆)alkenyl-COOH,             —NH(C₁₋₆)alkyl-(C₆₋₁₀)aryl-O(C₁₋₆)alkyl COOH,             —NH(C₁₋₆)alkyl-(C₆₋₁₀)aryl-COOH, —NHCH₂COOH, —NHCONH₂,             —NHCO(C₁₋₆)hydroxyalkyl COOH, —OCO(C₁₋₆)hydroxyalkyl COOH,             (C₃₋₆)cycloalkyl COOH,

—NHCN, —NHCHO, —NHSO₂CH₃, —NHSO₂CF₃; and —O(C₁₋₆alkyl)-tetrazol;

-   or R^(5a) is

-   wherein R⁷ and R⁸ are each independently H, (C₁₋₆ alkyl), (C₃₋₇     cycloalkyl), (C₁₋₆ alkyl)phenyl, (C₁₋₆ alkyl)-(C₃₋₇ cycloalkyl),     (C₃₋₇ cycloalkyl)-(C₁₋₆ alkyl), (C₃₋₇ cycloalkyl)-(C₂₋₄ alkenyl),     (C₁₋₆ alkyl)-OH, phenyl, CH₂biphenyl, 5- or 6-membered heterocycle     having from 1 to 4 heteroatoms selected from O, N, and S, 9- or     10-membered heterobicycle having 1 to 4 heteroatoms selected from O,     N, and S, (C₁₋₆ alkyl)-5- or 6-membered heterocycle having from 1 to     4 heteroatoms selected from O, N, and S, or (C₁₋₆ alkyl)-9- or     10-membered heterobicycle having 1 to 4 heteroatoms selected from O,     N, and S, or R⁷ and R⁸ are covalently bonded together to form (C₃₋₇     cycloalkyl), 4-, 5- or 6-membered heterocycle having from 1 to 4     heteroatoms selected from O, N, and S; -   or one of R⁷ or R⁸ is covalently bonded to R⁹ to form a pyrrolidine;     -   wherein said alkyl, cycloalkyl, heterocycle, heterobicycle,         phenyl are optionally substituted with from 1 to 4 substituents         selected from the group consisting of: OH, COOH, (C₁₋₆ alkyl),         (C₂₋₄ alkenyl), CONH₂, NH₂, NH(C₁₋₆ alkyl), N(C₁₋₆ alkyl)₂,         NHCOCOOH, NHCOCON(C₁₋₆ alkyl)₂, NHCOCONH(C₁₋₆ alkyl), SH, S(C₁₋₆         alkyl), NHC(═NH)NH₂, halogen, and COO(C₁₋₆alkyl); -   R⁹ is H or (C₁₋₆ alkyl); and -   Q is selected from the group consisting of: (C₁₋₃alkyl)CONHaryl, 6-     or 10-membered aryl, biphenyl, 5- or 6-atom heterocycle having 1 to     4 heteroatoms selected from O, N and S, 9- or 10-membered     heterobicycle having 1 to 4 heteroatoms selected from O, N and S;     -   wherein said aryl, biphenyl, heterocycle and heterobicycle are         all optionally substituted with from 1 to 4 substituents         selected from: OH, COOH, COO(C₁₋₆)alkyl, (C₁₋₆)alkyl,         (C₁₋₆)alkylCOOH, (C₁₋₆ alkyl)(C₂₋₄ alkynyl),         (C₁₋₆)alkyl-hydroxy, phenyl, benzyloxy, halogen, (C₂₋₄)alkenyl,         (C₂₋₄)alkenyl-(C₁₋₆)alkyl-COOH, 5- or 6-membered second         heterocycle having 1 to 4 heteroatoms selected from O, N and S,         NH-5- or 6- membered second heterocycle having 1 to 4         heteroatoms selected from O, N, and S,         -   wherein said second heterocycle and phenyl being optionally             substituted with from 1 to 4 substituents selected from:             (C₁₋₆ alkyl), CF₃, OH, (C₁₋₆alkyl) COOH, O(C₁₋₆alkyl)COOH,             (C₁₋₆alkyl) COO(C₁₋₆alkyl), CH₂phenyl, COO(C₁₋₆ alkyl),             (C₁₋₆alkyl)O(C₁₋₆alkyl), COOH, NCH(C₁₋₆alkyl)₂, NCO(C₁₋₆             alkyl), NH₂, NH(C₁₋₆ alkyl), halogen, N(C₁₋₆alkyl)₂; and             C₂₋₆ alkenyl-COOH     -   halogen, OPO₃H, benzyl, sulfonamido, SH, SOCH₃, SO₃H, SO₂CH₃,         S(C₁₋₆ alkyl)COOH, —CONH₂, —COCH₃, (C₁₋₃)alkyl,         (C₂₋₄alkenyl)COOH         -   wherein said alkenyl is optionally substituted with from 1             to 2 (C₁₋₆ alkyl) substituents,     -   (C₂₋₄alkenyl)COO(C₁₋₆alkyl), tetrazolyl, COOH, triazolyl, OH,         NO₂, NH₂, —O(C₁₋₆ alkyl)COOH, hydantoin, benzoyleneurea,         (C₁₋₄)alkoxy, (C₁₋₄)alkoxy(C₁₋₆ alkyl)COOH, cyano, azido,         —O—(C₁₋₆)alkyl COOH, —O—(C₁₋₆)alkyl COO—(C₁₋₆)alkyl, —NHCOCOOH,         —NHCOCONHOH , —NHCOCONH₂, —NHCOCONHCH₃, —NHCO(C₁₋₆)alkyl-COOH,         —NHCOCONH(C₁₋₆)alkyl-COOH, —NHCO(C₃₋₇)cycloalkyl-COOH,         —NHCONH(C₆₋₁₀)aryl-COOH, —NHCONH(C₆₋₁₀)aryl-COO(C₁₋₆)alkyl,         —NHCONH(C₁₋₆)alkyl-COOH, —NHCONH(C₁₋₆)alkyl-COO(C₁₋₆)alkyl,         —NHCONH(C₁₋₆)alkyl-(C₂₋₆)alkenyl-COOH,         —NH(C₁₋₆)alkyl-(C₆₋₁₀)aryl-O(C₁₋₆)alkyl COOH,         —NH(C₁₋₆)alkyl-(C₆₋₁₀)aryl-COOH, —NHCH₂COOH, —NHCONH₂,         —NHCO(C₁₋₆)hydroxyalkyl COOH, —OCO(C₁₋₆)hydroxyalkyl COOH,         (C₃₋₆)cycloalkyl COOH,

—NHCN, —NHCHO, —NHSO₂CH₃, —NHSO₂CF₃, coumarin, (C₁₋₆)alkyl-amino, NH(C₁₋₆alkyl)₂, C(halogen)₃, —NH(C₂₋₄)acyl, —NH(C₆₋₁₀)aroyl, —CON H(C₁₋₆-alkyl), —CO(C₁₋₆)alkyl-COOH, —CON H(C₁₋₆)alkyl-COOH, —CO—N H-alanyl, —CONH(C₂₋₄)alkylN(C₁₋₆alkyl)₂, —CONH(C₂₋₄)alkyl-Het, —CONH(C₂₋₄)alkyl-(COOH)-Het, —CONH(C₁₋₂ alkyl) (OH)(C₁₋₂ alkyl)OH, —CONH(C₁₋₆)alkyl-COOH, —CONH(C₆₋₁₀ aryl), —CONH-Het, —CONH(C₆₋₁₀)aryl-COOH, —CONH(C₆₋₀)aryl-COO(C₁₋₆)alkyl, —CONH(C₁₋₆)alkyl-COO(C₁₋₆)alkyl, —CONH(C₆₋₁₀)aryl-(C₁₋₆)alkyl-COOH, and —CONH(C₆₋₁₀)aryl-(C₂₋₆)alkenyl-COOH; or a salt thereof; said probe comprises a detectable label, whereby said probe binds to an HCV polymerase or an analog thereof and is capable of being displaced by an inhibitor thereof.

Labels incorporated into the probe may be paired with appropriate labels attached to the tagged NS5B polymerase such that the close proximity of the two pairs of labels upon probe-polymerase association results in a measurable signal; examples of such detection techniques include, but are not limited to, fluorescence resonance energy transfer (FRET), and time resolved fluorescence (TRF).

Preferably, the detectable label is selected from the group consisting of: a fluorescent label (such as fluorescein, Oregon green, dansyl, rhodamine, Texas-red, phycoerythrin or Eu^(3═)), a radioactive atom (such as ³H, ¹⁴C, ¹²⁵I), a chemiluminescent label (such as luciferase), colorimetric produced by an enzymatic marker (such as β-galactosidase or horseradish peroxidase).

Alternatively, a fluorescent reporter and quencher may be used as pair of labels to monitor association of the probe with the HCV NS5B polymerase. Commonly known reporter/quencher pair may be selected from, for example: EDANS/DABCYL, tryptophan/2,4-dinitrophenyl, tryptophan/DANSYL, 7-methoxycoumarin/2,4-dinitrophenyl, 2-aminobenzoyl/2,4-dinitrophenyl and 2-aminobenzoyl/3-nitrotyrosine.

As will be readily understood by a person skilled in the art, a radioactive label can be incorporated within the probe of formula I at any suitable position. For example, a ³H, or ¹⁴C isotope can replace any hydrogen or carbon present in the molecule. Similarly, a ¹²⁵I isotope can be substituted on any aromatic ring.

In principle, these tracer methodologies can easily be adapted for the purpose of high-volume screening. Scintillation proximity assay (SPA) methods for radioactive detection have been developed which do not require a separation step and are easily adapted for robotics and microtiter plate format.

Preferably, the detectable label is a fluorescent label or a chemiluminescent label. More preferably, the label is a fluorescent label. Most preferably, the detectable label is a fluorescein.

Non-radioactive detection methods have become increasingly widespread in screening assay because of the costs associated with radiolabeled reagents and their disposal. Fluorescence spectroscopy is one of the most prevalent non-radioactive detection methods. One type of assay in which fluorescence may be used is fluorescence polarization. Polarization is independent of total fluorescence intensity; therefore, this technique may not be as prone to interference as fluorescence amplitude measurements. As disclosed herein, the new type of assay developed uses a fluorescein-labeled inhibitor, though other fluorescent labels or non-fluorescent techniques can also be applied.

Preferably, the polymerase used in the assay may comprise an affinity tag by which the polymerase can be attached to a solid support, and the probe may be labeled so as to provide a detectable signal. An affinity tag incorporated into the probe maybe a biotin that is used to indirectly measure the association of this biotinylated probe to the NS5B polymerase through the secondary use of an avidin-coupled detection technique.

Preferably, the HCV polymerase used in the present assay is selected from the group consisting of: NS5B; NS5BΔ21; NS5BΔ57 or analogs thereof from a variety of genotypes including HCV-1a or 1b strains having optionally a histidine tag at either the N- or C-terminal. Particularly, as will be understood by a person skilled in the art, this binding assay does not require the polymerase activity of the NS5B to be optimal or functional for such a binding assay to perform according to the invention.

EXAMPLES

Probes of formula (I) can generally be synthesized according to the methods described in US 60/216,084; 60/274,374; 60/281,343; PCT CA01/00989; 13/089 filed Jul. 20, 2001 Pierre Louis Beaulieu, Guirez Fazal, George Kukolj, Martin Poirier and Youla Tsantrizos; and 13/090 filed Jul. 25, 2001 Pierre Louis Beaulieu, Guirez Fazal, Sylvie Goulet, George Kukolj, Martin Poirier, Youla Tsantrizos, Eric Jolicoeur, James Gillard and March-Andre Poupart herein incorporated by reference.

Example 1A probe (iii)): (S)-3-(5-Carboxymethoxy-1H-indol-3-yl)-2-({1-[1-cyclohexyl-2-(4-{[2-(5-dimethylamino-naphthalene-1-sulfonylamino)-ethylcarbamoyl]-methoxy}-phenyl)-1H-benzimidazol-5-yl]-methanoyl}-amino)-propionic acid

-   a) 4-Chloro-3-nitrobenzoic acid (40.40 g, 0.20 mole) was suspended     in DCM (100 mL) containing 3 drops of DMF. Oxalyl chloride (1.5     equivalents, 0.3 mole, 27 mL) was added in small portions and the     mixture stirred overnight at room temperature. After refluxing for     an additional hour to complete the reaction, volatiles were removed     under reduced pressure and the residue was co-evaporated twice with     hexane to give the title compound as a light yellow solid. -   b) (S)-5-Hydroxytryptophan methyl ester hydrochloride (1.55 g, 5     mmol) was dissolved in 80% aqueous MeCN (25 mL) and the solution     cooled in ice. Sodium bicarbonate (0.850 g, 10 mmol) was added     followed by di-telf-butyldicarbonate (1.10 g, 5.1 mmol). The mixture     was stirred for 2 h at room temperature, poured into water (200 mL)     and extracted with EtOAc (3×). The combined extracts were washed     with water and brine, dried (MgSO₄) and concentrated to give a beige     solid (1.65 g).

The crude product from above (1.50 g, 4.83 mmol) was dissolved in acetone (20 mL) and anhydrous potassium carbonate (1.5 g, 11 mmol) and methyl bromoacetate (0.76 g, 5 mmol) were added. The mixture was reflux for 4 h after which point additional methyl bromoacetate was added to complete the reaction (15 mg portions until complete by HPLC). The reaction mixture was then cooled and filtered to remove solid. Evaporation of the filtrate gave the desired carbamate as an oil (2.0 g).

The crude carbamate from above (2.0 g) was deprotected by stirring with 4N HCl—dioxane for 1 h at room temperature. Removal of volatiles in vacuo gave the desired tryptophan ester derivative as a tan-colored solid (1.51 g).

-   c) The tryptophan derivative from step b) (0.343 g, 1 mmol) was     dissolved in 80% aqueous MeCN (10 mL) and sodium bicarbonate (3     equivalents, 0.260 g) was added. The solution was cooled in ice and     4-chloro-3-nitrobenzoyl chloride from step a) (0.220 g, 1 mmol) was     added. The mixture was stirred for one hour at room temperature,     concentrated under reduced pressure and the residue purified by     flash chromatography (1:2 hexane/EtOAc as eluent) to give     compound c) as a yellow foam (0.391 g). -   d) The 4-chlorobenzamide derivative from above (0.214 g, 0.45 mmol)     was dissolved in DMSO (1 mL) and DIEA (0.2 mL) was added followed by     cyclohexylamine (3 equivalents, 0.16 mL). The mixture was stirred at     60-65° C. for 4 h and subsequently diluted with water. The orange     precipitate that formed was collected, washed with water and dried     (0.200 g). -   e) The crude material from above (0.200 g, 0.36 mmol) was     hydrogenated (1 atm H₂) over 20% Pd(OH)₂ on charcoal (60 mg) in MeOH     (15 mL). After 2 h, the suspension was filtered to remove the     catalyst and concentrated in vacuo to give the title compound as a     foam (0.16 g). -   f) 4-Formylphenoxyacetic acid (0.306 g, 1.70 mmol) was dissolved in     DCM (5 mL). DIEA (0.524 g, 4 mmol) and TBTU (0.550 g, 1.70 mmol)     were added followed by tert-butyl N-(2-aminoethyl)carbamate (0.250     g, 1.56 mmol). The mixture was stirred 2 h at room temperature,     dissolved in EtOAc and washed sequentially with 5% aqueous K₂CO₃,     KHSO₄, water and brine. The extract was dried (MgSO₄) and     concentrated under reduced pressure to give a yellow solid (0.350     g). -   g) The diamine derivative from step e) (0.026 g, 0.05 mmol) and     aldehyde from step f) (0.020 g, 0.06 mmol) were dissolved in DMF     (0.3 mL) and water (0.03 mL) was added followed by oxone®) (0.024 g,     0.04 mmol). The mixture was stirred 1 h at room temperature and then     diluted with water. The resulting precipitate was collected by     filtration, washed with water and dried to give a beige solid (0.020     g). -   h) The crude carbamate from above was stirred with TFA for 30 min at     room temperature. Volatiles were removed under reduced pressure and     the residue was purified by preparative C₁₋₈ reversed-phase HPLC to     give the bis TFA salt. -   i) The amine salt (0.019 g, 0.02 mmol) was dissolved in DMSO (0.3     mL) and DIEA (0.06 mL) was added followed by dansyl chloride (0.065     g, 0.02 mmol). The mixture was stirred for 1 h at room temperature.     5N NaOH (0.12 mL) and water (0.05 mL) were added and the     saponification was allowed to proceed for 1 h at room temperature.     Following acidification with TFA, the probe (iii) was directly     isolated from the reaction mixture by preparative C₁₋₈     reversed-phase HPLC: MS (ES+) m/z 930 (MH+).

Example 1B probe (ii):5-(3-{2-[2-(4-{5-[(S)-1-Carboxy-2-(5-carboxymethoxy-1-H-indol-3-yl)-ethylcarbamoyl]-1-cyclohexyl-1H-benzimidazol-2-yl}-phenoxy)-ethanoylamino]-ethyl}-thioureido)-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)-benzoic acid

The amine salt from step h) of Example 1A (0.06 mmol) was dissolved in DMSO (0.6 mL) and DIEA (0.3 mL) was added followed by fluorescein isothiocyanate isomer 1 (0.026 g, 0.066 mmol). The mixture was stirred for 1 h at room temperature. 5N NaOH (0.3 mL) and water (0.15 mL) were added and stirring resumed for an additional 30 min. Following acidification with TFA, probe (ii) was isolated directly by preparative C₁₋₈ reversed-phase HPLC: MS (ES+) m/z 1086 (MH+).

Example 1C probe (v): (S)-2-{[1-(2-{4-[(2-{[1-(4-Azido-phenyl)-methanoyl]-amino}-ethylcarbamoyl)-methoxy]-phenyl}-1-cyclohexyl-1H-benzoimidazol-5-yl)-methanoyl]-amino}-3-(5-carboxymethoxy-1H-indol-3-yl)-propionic acid

-   a) 4-Azidobenzoic acid (0.160 g, 1 mmol) was dissolved in DCM (3     mL). DIEA (0.5 mL, 2.5 mmol) and TBTU (0.337 g, 1.05 mmol) were     added followed by tert-butyl N-(2-aminoethyl)carbamate (0.165 g,     1.03 mmol). The mixture was stirred 2.5 h at room temperature,     dissolved in EtOAc and washed sequentially with 5% aqueous K₂CO₃,     KHSO₄, water and brine. The extract was dried (MgSO₄) and     concentrated under reduced pressure to give a yellow solid (0.257     g). The crude carbamate (0.257 g, 0.84 mmol) was deprotected by     stirring in 4N HCl—dioxane (15 mL) for 2 h at room temperature.     Volatiles were removed under reduced pressure to give a pinkish     solid. -   b) 4-Formylphenoxyacetic acid (0.200 g, 1.1 mmol) was dissolved in     DCM (3 mL) and DIEA (0.5 mL) was added followed by TBTU (0.350 g,     1,1 mmol) and the amine salt from above (0.240 g, 1 mmol). The     mixture was stirred 4 h at room temperature, dissolved in EtOAc and     washed sequentially with 5% aqueous K₂CO₃, KHSO₄, water and brine.     The extract was dried (MgSO₄) and concentrated under reduced     pressure to give an off-white solid (0.162 g). -   c) The benzaldehyde derivative from above (0.044 g, 0.12 mmol) and     the diamine derivative from step e) of Example 1A (0.052 g, 0.1     mmol) were dissolved in DMF (0.6 mL) and water (0.1 mL). Oxone®)     (0.050 g, 0.8 mmol) was added and the mixture stirred for 1 h at     room temperature. 5N NaOH (0.2 mL) and water (0.1 mL) were added and     saponification allowed to proceed for 1 h. Probe (v) was isolated     directly by preparative C₁₋₈ reversed-phase HPLC (12.5 mg): MS (ES+)     m/z 842 (MH+).

Example 1D probe (vi): (S)-3-(5-Carboxymethoxy-1H-indol-3-yl)-2-{(1-[1-cyclohexyl-2-(4-{[2-({1-[4-(1-phenyl-methanoyl)-phenyl]-methanoyl}-amino)-ethylcarbamoyl]-methoxy}-phenyl)-1H-benzoimidazol-5-yl]-methanoyl}-amino)-propionic acid

Following the procedures described for probe (v) in Example 1C but using 4-benzoylbenzoic acid instead of 4-azidobenzoic acid, probe (vi) was obtained: MS (ES+) m/z 905 (MH+).

Example 1E Probe (iv) (S)-3-(5-Carboxymethoxy-1H-indol-3-yl)-2-{[1-(1-cyclohexyl-2-{4-[2-(5-dimethylamino-naphthalene-1-sulfonylamino)-ethylcarbamoyl]-phenyl}- 1H-benzimidazol-5-yl)-methanoyl]-amino}-propionic acid

-   a) Following the procedures described for step f) in Example 1A,     4-carboxybenzaldehyde was coupled to tert-butyl     N-(2-aminoethyl)carbamate. -   b) Following benzimidazole ring formation with the diamine     derivative of Example 1A step e) and the aldehyde from above using     oxone® as described in Example 1A step g), the Boc protecting group     was removed and the resulting amine condensed with dansyl chloride     as described in Example 1A step i). -   c) Probe (iv) was obtained following saponification of the ester     groups under the usual conditions and isolation by preparative C₁₋₈     reversed-phase HPLC: MS (ES+) m/z 900 (MH+).

Example 1F (Probe (i):5-[3-(2-{[1-(4-{5-[(S)-1-Carboxy-2-(5-carboxymethoxy-1-H-indol-3-yl)-ethylcarbamoyl]-1-cyclohexyl-1H-benzimidazol-2-yl}-phenyl)-methanoyl]-amino}-ethyl)-thioureido]-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)-benzoic acid

The procedure described for Example 1E) was used except that fluorescein isothiocyanate isomer 1 was used instead of dansyl chloride. Probe (i) was obtained after purification by preparative C₁₋₈ reversed-phase HPLC: MS (ES+) m/z 1056 (MH+).

Example 2 Production and Purification of HCV NS5B polymerase Δ21-His

The recombinant HCV NS5B polymerase can be produced in soluble form by expression of a variant that lacks the C-terminal 21 amino acids normally found on the mature NS5B (Yamashita et al. 1998, J. Biol. Chem. 273:15479-15486; Ferrari et al., 1999, J. Virol. 73: 1649-1654). We have expressed this so called NS5BΔ21 with a C-terminal hexa-histidine (termed NS5BΔ21-His; SEQ ID. NO. 1) and with an N-terminal hexa-histidine tag (termed His-NS5BΔ21; SEQ ID NO. 2) (either proteins being referred to as “his-tag NS5B”). Expression of these genes from pET vectors in E. coli strain JM109 (DE3) is induced with 0.4 mM IPTG for 3 hours at 22° C. Cells are harvested and lysed in a microfluidizer in lysis buffer (Tris-HCl pH 7.5, 10% glycerol, 1 mM EDTA, 2 mM 2-mercaptoethanol, 500 mM NaCl, 1 mM PMSF, 1 μg/ml antipain, 1 μg/ml pepstatin A and 1 μg/ml leupeptin). The lysate is clarified by a 30 000 g centrifugation and then supplemented with imidazole to final concentration of 10 mM. The lysate is then loaded onto a metal-chelating resin (Ni-NTA; Qiagen) previously equilibrated with buffer A (Tris-HCl pH 7.5, 10% glycerol, 500 mM NaCl, 10 mM imidazole), washed extensively and then the protein is eluted with gradient of buffer A containing 500 mM imidazole. Peak fractions containing the his-tag NS5BΔ21 are pooled and diluted with buffer C (20 mM Tris-HCl pH 7.5, 10% glycerol, 5 mM DTT) to reduce the NaCl concentration to 300 mM and then applied to a DEAE-Sepharose column to remove any nucleic acid. The flow-through from the DEAE-Sepharose column is diluted with buffer C to reduce the NaCl to 200 mM and then applied to a heparin-Sepharose column. The his-tag NS5B is eluted from the heparin-Sepharose in buffer C with a 200 mM to 1 M NaCl gradient. Peak fractions containing the his-tag NS5B are pooled and diluted with buffer C to achieve a final NaCl of 200 mM and loaded onto a Resource S column. Concentrated His-tag NS5B is eluted from the resource S, loaded and size fractionated on a Superdex 200 column in buffer C containing 300 mM NaCl. Peak fractions contain highly pure his-tag NS5B and are stored at −80° C. until use.

Example 3 Fluorescence Anisotropy Analysis

Titration of the probe with the enzyme was performed as follows:

The fluorescein labeled probe was diluted to the desired concentration in 20 mM Tris-HCl pH 7.5, 1 mM EDTA, 5 mM MgCl₂, 1 mM DTT and 10% DMSO. The NS5BΔ21-His protein was serially diluted in 25 mM Tris-HCl pH 7.5, 300 mM NaCl, 5 mM DTT, 1 mM EDTA, 30% glycerol and 0.1% IGEPAL. Total volume of the reaction was 500 μL and final assay buffer was 20 mM Tris-HCl pH 7.5, 1 mM EDTA, 5 mM MgCl₂, 1 mM DTT, 30 mM NaCl, 3% glycerol, 0.01% IGEPAL and 5% DMSO. Anisotropy measurements were performed on a SLM Aminco 8100 Spectrofluorometer equipped with a 450-W xenon arc lamp and a T-optics configuration. Excitation wavelength was at 493 nm and emission was monitored at 530 nm. In each anisotropy measurement, the parallel and perpendicular intensities of the background buffer solution was subtracted from the measured values of the sample and the anisotropy was calculated. Data were processed on SAS program (SAS Institute Inc., NC, USA) for a non linear regression to obtain the direct binding equilibrium constant and other parameters, and the plot of the regressed fit over the experimental data. An example of a titration curve obtained with probe (i) is shown on FIG. 1. K_(d) values for probes (i) and (ii) with the polymerase were respectively of 15 nM and 6 nM.

Example 4 96-Well Plate Polarization Assay

To obtain K_(d) values of different compounds competing with these probes (test compounds), this assay was transformed to a more amenable format and a binding assay was made suitable for a 96-well microplate reader. The probe was diluted in order to obtain the desired final concentration (from 4 to 25 nM, depending on its K_(d) towards the enzyme and on the conditions of the assay). The tested compounds were serially diluted by a factor of 2 or 3-fold in 20 mM Tris-HCL pH 7.5, 1 mM EDTA, 5 mM MgCl₂, 1 mM DTT and 15% DMSO. The NS5BΔ21-His concentration in the assay was calculated to obtain 70% of binding of the probe; these conditions allowed for the displacement of the probe by test compounds. The assay reactions finally contained 50 μL of the serial dilutions of the tested compounds that were transferred in 96-well black plates (Packard); a complete row was however free of compound to obtain a positive control value and verify real percent of bound probe in the experiment. 50 μL of the probe were then added to each well, except in one column for blank subtraction. Lastly, 150 μL of enzyme were added to all wells, except in one row, which was used to determine the 0% and 100% bound values. In this row of 8 wells, enzyme buffer was added to the first 4 wells (to determine the anisotropy value of the free probe or r_(f)) and a 10-fold excess of the concentration of the enzyme used in the assay was added to the other 4 wells (to determine the anisotropy value when 100% of the probe is bound i.e. the r_(b) value). These values were required to calculate the K_(d) values. The final buffer conditions of the assay were identical to the ones used for K_(d) determination of the probes, i.e. 20 mM Tris-HCl pH 7.5, 1 mM EDTA, 5 mM MgCl₂, 1 mM DTT, 30 mM NaCl, 3% glycerol, 0.01% IGEPAL and 5% DMSO. The reactions were incubated for 90 minutes at room temperature in the dark. Readings of polarization were then performed on a POLARstar Galaxy, equipped with a high-energy xenon flash lamp, using an excitation filter of 485 nm and an emission filter of 520 nm. Polarization values can be converted easily to anisotropy values with the following calculation (Owicki et al., 2000, J. Biomol. Screen. 5:297-306): a=2×P/(3−P) where

-   a: anisotropy value -   P: Polarization value

Anisotropy values can then be used to obtain two types of results fitted to SAS nonlinear regression analysis to obtain apparent K_(d) values, using for the calculations as positive control the anisotropy value at ˜70% binding, and as negative control the anisotropy value of the free probe (r_(f)); fitted to the Anisotropy equation:

$a = \frac{\left( {{- K_{d}} - I + E_{o}} \right) + \sqrt{\left( {\left( {K_{d} + I - E_{o}} \right)^{2} + {4*K_{d}*E_{o}}} \right)}}{2\left\lbrack \frac{\left( {{a*Q*r_{b}} + {K_{p}*r_{f}}} \right)}{\left( {K_{p} + {a*Q}} \right)} \right\rbrack}$ where a: anisotropy

-   -   K_(d): dissociation constant for the inhibitor     -   I: Concentration of compound (or inhibitor) tested     -   E_(o): NS5B concentration (E_(o) has to be >>[probe])     -   Q=Q_(b)/Q_(f)=total fluorescence for probe 100% bound/total         fluorescence for free probe     -   r_(b): anisotropy value when the probe is 100% bound     -   r_(f): anisotropy value when the probe is free     -   K_(p): dissociation constant for the probe

This high throughput assay was evaluated and validated by the determination of the statistical parameter Z′ (J.-H. Zhang et al., 1999, J. of Biomol. Screening, 4:67-73). Results of this experiment are illustrated on FIG. 2. The anisotropy values for a series of positive and negative controls were very similar, resulting in very low standard deviations; 0.2186±0.0036 A units for the positive controls and 0.0738±0.0037 A units for the negative controls. The Z′ value obtained for the assay was of 0.85, implying that we have excellent conditions to detect compounds that would compete with the probe.

Example 5 Inhibitor Testing

We have identified potent compounds that can effectively displace the probe in this binding assay. FIGS. 3 and 4 show examples of some of them, with K_(d) values ranging from 31 nM to 1 μM. The anisotropy equation was defined in the Grafit Software (Erithacus Software Ltd., UK) and plotted such that inhibitor concentration was the X-variable and anisotropy was the Y variable; parameters calculated by the software were the inhibitor K_(d) and Q_(b)/Q_(f) ratio. Supplied constants were the K_(p), E_(O), r_(b) and r_(f).

Example 6 Modified Conditions for the Polarization Assay

The usefulness of this polarization assay is evident when binding of compounds has to be studied under different conditions. For example, binding constants of the probes have been determined at different concentrations of salts and pH. FIGS. 5 to 8 show the binding curves of probe (i) in final NaCl concentrations ranging from 30 mM to 200 mM. FIG. 5 shows the NaCl concentration at 30 mM. FIG. 6 shows the NaCl concentration at 100 mM. FIG. 7 shows the NaCl concentration at 150 mM. FIG. 8 shows the NaCl concentration at 200 mM. All other reagents in the assay were as described in the standard protocol (Example 3). As shown on these Figures, K_(d) values gradually increase with salt concentration from K_(d)=15 nM (at 30 mM NaCl) to K_(d)=122 nM (at 200 mM NaCl). Studies at pH 6.5 were also performed to determine the K_(d) of the probe (i) at lower pH. For these assays, 20 mM Phosphate buffer pH 6.5 was used in place of Tris; all other reagents of the assay were as described in the 96-well Polarization assay (Example 4). An example of these types of experiments is shown in FIG. 9. The K_(d) value obtained at pH 6.5 with probe (i) was of 33 nM. Having established these K_(d) values under different experimental conditions, it is then trivial to determine what concentrations of probe and enzyme should be used to obtain 70% of binding of the probe with the equilibrium equation. Once these values are obtained, compounds of interest can easily be studied under the new conditions to determine their K_(d) values.

Example 7 Fluorescence Polarization Assay With a Modified Enzyme

The Fluorescence polarization assay was also used with other constructs of our HCV polymerase enzyme. In addition to the C-terminally tagged NS5BΔ21-His polymerase, the NS5B enzyme with the His-tag at the N-terminal position was also used in the fluorescence polarization assay. Determination of the K_(d) for the probe (i) with this enzyme was performed, using the same conditions described in the standard 96-well format assay. FIG. 10 shows that the K_(d) obtained with probe (i) was similar, i.e. 18 nM. A comparison was made between the IC₅₀ and the K_(d) for three compounds, using these two different constructs of the enzymes (NS5BΔ21-His and His-NS5BΔ21).

IC₅₀'s are determined using the Scintillation Proximity Assay (SPA) according to the following assay:

The substrates are: (i) a 12 nucleotide RNA oligo-uridylate (or oligo-uridine-monophosphate) (oligo-U) primer modified with biotin at the free 5° C. position; (ii) a complementary poly-adenylate (or adenosine monophospahte) (polyA) template of heterogeneous length (1000-10000 nucleotides); and (iii) UTP-[5,6 ³H]. Polymerase activity is measured as the incorporation of UMP-[5,6 ³H] into the chain elongated from the oligo-U primer. The ³H-labelled reaction product is captured by SPA-beads coated with streptavidin and quantified on the TopCount (Packard). Inhibitors are tested at various concentrations in a reaction containing: 1 to 5 nM of the his-tagged NS5B, 1 μg/ml of biotinylated oligo U primer, 10 μg/ml of polyA template, 20 mM Tris-HCl pH 7.5, 5 mM MgCl₂, 25 mM KCl, 1 mM EDTA, 1 mM DTT, 0.33% n-dodecyl maltoside, 5% DMSO, 0.0083 μCi/μl [0.25 μM] UTP-[5,6-³H], 0.75 μUTP, 1.67 μl RNAsin™. The reaction was incubated at room temperature for 1.5 hours. STOP solution (20 μl; 0.5 M EDTA, 150 ng/μl tRNA) was added, followed by 30 μl streptavidin coated PVT beads (8 mg/ml in 20 mM Tris-HCl, pH 7.5, 25 mM KCl, 0.025% NaN₃). The plate was then shaken for 30 minutes. A solution of CsCl was added (70 μl, 5 M), to bring the CsCl concentration to 1.95 M. The mixture was then allowed to stand for 1 hour. The beads were then counted on a Hewlett Packard TopCount™ instrument. Based on the results at ten different concentrations of test compound, standard concentration-% inhibition curves were plotted and analysed to determine IC₅₀'s for the compounds.

Results of this experiment are illustrated in Table I. The K_(d) values were similar with both enzymes for the three compounds tested, whereas the IC₅₀ values obtained with the two enzymes show significant differences and reflect the differences in substrate affinity.

Example 8 Specificity of the Fluorescence Polarization Assay

The utility of the Fluorescence polarization assay was examined with another distantly related viral polymerase and with a closely related genotype (1a) HCV polymerase.

The GBV-B polymerase enzyme (termed GBV-BΔ23-His; SEQ ID NO. 3) (Simons, J. N. et al., 1995, Proc. Natl. Acad. Sci. USA 92, 3401-3405; Bukh, J. et al., 1999, Virology 262, 470-478) was produced and purified as described in Example 2 with the following modifications:

Expression of the gene from pET vectors in E. coli strain JM109 (DE3) was induced with 0.5 mM IPTG for 3 hours at 22° C. Cells were harvested and lysed in a microfluidizer in buffer A (Tris-HCl pH 7.5, 10% glycerol, 1 mM EDTA, 2 mM 2-mercaptoethanol, 500 mM NaCl, 1 mM PMSF, 1 ug/ml antipain, 1 ug/ml pepstatin A, 1 ug/ml leupeptin and 0.5% dodecyl-β-D-maltoside). The lysate was clarified by a 30 000 g centrifugation and then supplemented with imidazole to a final concentration of 10 mM. The lysate was then loaded onto a metal-chelating resin (Ni-NTA; Qiagen) previously equilibrated with buffer A containing 10 mM imidazole, washed extensively and then the protein was eluted with a gradient of buffer A containing 500 mM imidazole. Peak fractions containing the his-tag GBV-BΔ23 were pooled and diluted with buffer C (20 mM Tris-HCl pH 7.5, 10% glycerol, 5 mM DTT, 0.01% dodecyl-β-D-maltoside) to reduce the NaCl concentration to 300 mM and then applied to a DEAE-Speharose column to remove any nucleic acid. The flow-through from the DEAE-Speharose column was diluted with buffer C to reduce the NaCl to 200 mM and then applied to a heparin-Sepharose column. The his-tag GBV-B was eluted from the heparin-Sepharose in buffer C with a 200 mM to 1 M NaCl gradient. Peak fractions containing the pure his-tag GBV-B were then pooled and stored at −80° C. until use.

The HCV genotype 1a NS5B polymerase [termed His-NS5BΔ21 (H77c, 1a); SEQ ID NO. 4] (Yanagi, M. et al., 1997, Proc. Natl. Acad. Sci. USA 94, 8738-8743) was produced and purified as described in Example 2 with the following modifications:

Expression of the gene from pET vectors in E. coli strain JM109 (DE3) was induced with 0.4 mM IPTG for 3 hours at 22° C. Cells were harvested and lysed in a microfluidizer in buffer A (Tris-HCl pH 8.0, 10% glycerol, 1 mM EDTA, 2 mM 2-mercaptoethanol, 500 mM NaCl, 1 mM PMSF, 1 ug/ml antipain, 1 ug/ml pepstatin A, 1 ug/ml leupeptin, 1% dodecyl-β-D-maltoside, 1% Triton X-100 and 0.1% CHAPS). The lysate was clarified by a 30 000 g centrifugation and then supplemented with imidazole to a final concentration of 10 mM. The lysate was then loaded onto a metal-chelating resin (Ni-NTA; Qiagen) previously equilibrated with buffer A containing 10 mM imidazole, 0.1% NP-40, without CHAPS, and with lower concentrations of the other detergents (0.2% dodecyl-β-D-maltoside, 0.05% Triton X-100); after extensive washing, the protein was eluted with a gradient of buffer A containing 500 mM imidazole. Peak fractions containing the his-tag NS5BΔ21 (H77c, 1a) were pooled and diluted with buffer C (20 mM Tris-HCl pH 8.0, 10% glycerol, 5 mM DTT, 0.2% dodecyl-β-D-maltoside) to reduce the NaCl concentration to 300 mM and then applied to a DEAE-Sepharose column to remove any nucleic acid. The flow-through from the DEAE-Sepharose column was diluted with buffer C to reduce the NaCl to 200 mM and then applied to a heparin-Sepharose column. The his-tag NS5BΔ21(H77c, 1a) was eluted from the heparin-Sepharose in buffer C with a 200 mM to 1 M NaCl gradient. Peak fractions containing the polymerase were then pooled and diluted with buffer C to achieve a final NaCl of 200 mM and loaded onto a Resource S column. Peak fractions containing the his-tag NS5B(H77c, 1a) were pooled, loaded and size fractionated on a Superose 12 column in buffer C containing 600 mM NaCl. Peak fractions contain highly pure his-tag NS5B were pooled and stored at −80° C. until use.

The GBV-B and the HCV 1a polymerases were used to titrate probe ii, using the protocol described in Example 3. FIGS. 11 and 12 show the titration curves observed with the GBV-B polymerase and the NS5B(H77c, 1a) polymerase, respectively. The K_(d) value of probe ii for the GBV-B enzyme was 1.8 uM (estimated value with an incomplete curve and an r_(b) value of 0.21), illustrating the weak binding of the probe to this distantly related polymerase. In contrast, the K_(d) for the HCV 1a polymerase was 18 nM, revealing that the 1a genotype enzyme binds probe ii with the same affinity as the HCV 1b genotype polymerase. K_(d) values for a series of compounds were determined with these two HCV (genotypes 1a and 1b) polymerases, using the assay format described in Example 4.

Results of this experiment are illustrated in Table 2. These results show that the K_(d) values for this series of inhibitors are in the same range with the two genotypically related HCV enzymes.

TABLE 1 Comparison of compound K_(d) and IC₅₀ values with two different HCV NS5B polymerases K_(d) value (nM) His- IC₅₀ value (nM) Cpd NS5BΔ21-His NS5BΔ21 NS5BΔ21-His His-NS5BΔ21 X 44 41 867 66 Y 22 31 348 68 Z 92 88 735 34

TABLE 2 Comparison of compound K_(d) values with NS5B polymerases from two HCV genotypes K_(d) values (nM) Cpd His-NS5BΔ21(1b) His-NS5BΔ21(H77c, 1a) A 2.7 1.8 B 12 8.0 C 5.3 7.2 D 3.5 7.1 E 2.4 2.7 Discussion

The HCV NS5B polymerase is a prime target in the search for inhibitors of HCV replication. The HCV NS5B enzymatic activity has been studied in vitro with a variety of RNA substrates (Behrens et al., 1996; and many references thereafter). Different preparations of the HCV polymerase exhibit varying efficiencies of product formation with a variety of RNA substrates. Estimations are that only a small fraction (i.e. <1%) of the common preparations of purified recombinant HCV NS5B polymerase interact with RNA substrate to reconstitute processive RNA product synthesis (Carroll SS, et al., 2000. Biochemistry, 39:8243-8249). Moreover, the activity of purified recombinant NS5B polymerase varies significantly with specific RNA substrates; a characteristic that presumably reflects the capability of the NS5B of forming productive replication-competent complexes with these substrates (Zhong W, et al., 2000, J Virol, 74, 9134-9143).

In an effort to overcome the limitations of HCV polymerase assays that use sub-optimal and poorly characterized RNA substrates, the Applicants have developed an assay for specific inhibitors of the HCV polymerase that is independent of the presence of RNA. The assay is based upon the use of a characterized inhibitor specific for the HCV polymerase. In the examples presented above, the inhibitor was labeled with a fluorescein moiety and the interaction of this probe with the NS5B was measured and quantified by fluorescence polarization. However, the interaction can also be measured by the use of a radiolabel, or other common labels placed on the inhibitor and applying common techniques for assessing the association of the labeled probe with an appropriately tagged target HCV polymerase. Binding equilibrium with the fluorescein labeled probe is clearly evident in Example 3, as the fraction of bound probe increased with the amount of HCV polymerase. An HCV polymerase assay with components at equilibrium is an advantage over previous assays with RNA substrates, as the active HCV polymerase that stably associates with RNA substrates in processive complexes does not readily dissociate (Carroll SS, et al., 2000 Biochemistry, 39:8243-8249; Zhong W, et al., 2000 J Virol, 74, 9134-9143; Tomei L, et al. 2000 J. Gen. Virol. 81, 759-767). Though these labeled probes readily dissociate from the HCV polymerase, they do so with low nM dissociation constants and provide the required sensitivity (in the low nM range) to detect potent and specific inhibitors. The assay format is adaptable to screening in 96-well (or higher density) plate format as demonstrated in Example 4. A particular advantage of this high throughput screening format is the extremely stable signal and minimal well-to-well variation that the assay provides, particularly in a convenient non-radioactive format. Specific inhibitors of the HCV polymerase were identified and potencies easily determined with this assay (FIGS. 3 and 4).

The direct binding assay described herein overcomes other limitations of the enzymatic HCV polymerase assay. The in vitro RNA polymerase activity of NS5B is extremely sensitive to ionic strength, and KCl or NaCl concentrations exceeding 100 mM inhibit the reaction (Lohmann V, et a., 1998 Virology 249,108-118; Luo G, et al., 2000, J. Virol., 74, 851-63.) Hence the ability to determine the potency of inhibitors at various salt concentrations is restricted by this limitation of standard enzymatic reactions. The direct binding assay of this invention is amenable to adjustments in salt concentration or pH levels as demonstrated in Example 6. The potencies and interaction of specific inhibitors with the NS5B target can easily be determined under conditions not suitable for enzymatic RNA polymerization studies (such as the absence of divalent cation).

Established HCV polymerase enzymatic assays provide IC₅₀ values as representative measurements of inhibitor potencies. For inhibitors that are competitive with either RNA or NTP, the IC₅₀ value is proportional to the concentration of substrates in the assay and will vary depending on the concentration of these components. The assay described herein permits a direct measurement of inhibitor potencies (reflected by K_(d) values), under defined conditions, irrespective of the substrate concentration. In enzymatic reactions that use either the N-terminal tag His-NS5BΔ21 or the C-terminal tag NS5BΔ21-His, significantly disparate IC₅₀ values are obtained for identical compounds assayed under identical conditions. The His-NS5BΔ21 and NS5BΔ21-His polymerases have different affinities for the primer/template RNA substrate thereby resulting in the disparate IC₅₀ for the identical compounds (Example 7, Table 1). A major advantage that is exemplified by the direct binding assay described in this invention is that these differences are reconciled by the relatively similar K_(d) values that the individual inhibitors display with the two different HCV polymerases.

The direct binding assay described herein has also been shown to be specific for HCV polymerase enzymes. Example 8, in which a K_(d) at least 100-fold higher for the probe ii was obtained with the GBV-B polymerase, illustrates the weak binding of the probe to this polymerase and the specificity of binding to the HCV polymerases. Moreover, Example 8 also demonstrates that the polymerases from two distinct and clinically relevant HCV genotypes bind the probe with similar affinities.

The direct inhibitor-binding assay of this invention alleviates many restrictions of conventional HCV polymerase enzymatic assays described to date. The Applicants have exemplified how the use of a characterized inhibitor as a competitive probe provides a number of improvements and advancements in the search for specific inhibitors of the NS5B polymerase. This assay may accelerate the identification and characterization of candidate therapeutics for the treatment of HCV related diseases.

All documents referred to herein are hereby incorporated by reference in their entirety. 

1. A method for identifying compounds binding to Hepatitis C Virus polymerase comprising the steps of: a) contacting said Hepatitis C Virus polymerase with a probe being capable of binding to said Hepatitis C Virus polymerase, so as to form a complex comprising said probe bound to said polymerase; b) measuring a signal emitted from said probe in said complex to establish a base line level: c) incubating said complex from step a) with a test compound; d) measuring said signal from said complex from step c); and e) comparing said signal from step d) with said signal from step b) wherein said probe is comprised of a compound according to general formula Ia:

wherein R² is CON(R²²)₂, wherein each R²² is independently H, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, (C₅₋₇)cycloalkenyl, 6 or 10-membered aryl or Het, or both R²² are bonded together to form a 5, 6 or 7-membered saturated heterocycle with the nitrogen to which they are attached; or R² is selected from: H, halogen, (C₁₋₆)alkyl, haloalkyl, (C₂₋₆)alkenyl, (C₅₋₇)cycloalkenyl, 6 or 10-membered aryl or Het; wherein each of said alkyl, haloalkyl, (C₂₋₆)alkenyl, (C₅₋₇)cycloalkenyl, aryl or Het is optionally substituted with R²⁰, wherein R²⁰ is defined as: 1 to 4 substituents selected from: halogen, NO₂, cyano, azido, C(═NH)NH₂, C(═NH)NH(C₁₋₆)alkyl or C(═NH)NHCO(C₁₋₆)alkyl; or 1 to 4 substituents selected from: a) (C₁₋₆)alkyl or haloalkyl, (C₃₋₇)cycloalkyl, (C₂₋₆)alkenyl, (C₂₋₈)alkynyl, (C₁₋₆) alkyl-(C₃₋₇)cycloalkyl, all of which optionally substituted with R¹⁵⁰; b) OR¹⁰⁴ wherein R¹⁰⁴ is H, (C₁₋₆alkyl), (C₃₋₇)cycloalkyl, or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, said alkyl, cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het being optionally substituted with R¹⁵⁰; c) OCOR¹⁰⁵ wherein R¹⁰⁵ is (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, said alkyl, cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het being optionally substituted with R¹⁵⁰; d) SR¹⁰⁸, SO₃H, SO₂N(R¹⁰⁸)₂ or SO₂N(R¹⁰⁸)C(O)R¹⁰⁸ wherein each R¹⁰⁸ is independently H, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆)Het or both R¹⁰⁸ are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het or heterocycle being optionally substituted with R¹⁵⁰; e) NR¹¹¹R¹¹² wherein R¹¹¹ is H, (C₁₋₆)alkyl, C(₃₋₇)cycloalkyl or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het and R¹¹² is H, CN, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl, (C₁₋₆alkyl)Het, COOR¹¹⁵ or SO₂R¹¹⁵ wherein R¹¹⁵ is (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, or both R¹¹¹ and R¹¹² are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, or heterocycle being optionally substituted with R¹⁵⁰; f) NR¹¹⁶COR¹¹⁷ wherein R¹¹⁶ and R¹¹⁷ is each H, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, said (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cyclocalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het being optionally substituted with R¹⁵⁰; g) NR¹¹⁸CONR¹¹⁹R¹²⁰, wherein R¹¹⁸, R¹¹⁹ and R¹²⁰ is each H, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, or R¹¹⁸ is covalently bonded to R¹¹⁹ and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle; or R¹¹⁹ and R¹²⁰ are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle; said alkyl, cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het or heterocycle being optionally substituted with R¹⁵⁰; h) NR¹²¹COCOR¹²² wherein R¹²¹ and R¹²² is each is H, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, a 6- or 10-membered aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, said alkyl, cycloalkyl, alkyl-cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het being optionally substituted with R¹⁵⁰; or R¹²² is OR¹²³ or N(R¹²⁴)₂ wherein R¹²³ and each R¹²⁴ is independently H, (C₁₋₆alkyl), (C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, or R¹²⁴ is OH or O (C₁₋₆alkyl) or both R¹²⁴ are covalently bonded together to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl, alkyl-cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het and heterocycle being optionally substituted with R¹⁵⁰; i) COR¹²⁷ wherein R¹²⁷ is H, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, said alkyl, cycloalkyl, aryl, Het (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het being optionallyl substituted with R¹⁵⁰; j) COOR¹²⁸ wherein R¹²⁸ is H, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, said (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl and (C₁₋₆alkyl) Het being optionally substituted with R¹⁵⁰; k) CONR¹²⁹R¹³⁰ wherein R¹²⁹ and R¹³⁰ are independently H, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, or both R¹²⁹ and R¹³⁰ are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl, alkyl-cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl, (C₁₋₆alkyl)Het and heterocycle being optionally substituted with R¹⁵⁰; l) aryl, Het, (C1-6alkyl)aryl or (C1-6alkyl)Het, all of which being optionally substituted with R¹⁵⁰; wherein R¹⁵⁰ is preferably: 1 to 3 substituents selected from: halogen, NO₂, cyano or azido; or 1 to 3 substituents selected from: a) (C₁₋₆)alkyl or haloalkyl, (C₃₋₇)cycloalkyl, (C₂₋₆)alkenyl, (C₂₋₈)alkynyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, all of which optionally substituted with R¹⁶⁰; b) OR¹⁰⁴ wherein R¹⁰⁴ is H, (C₁₋₆alkyl) or said alkyl or cycloalkyl optionally substituted with R¹⁶⁰; d) SR¹⁰⁸, SO₃H, SO₂N(R¹⁰⁸)C(O)R¹⁰⁸ wherein each R¹⁰⁸ is independently H, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, or both R¹⁰⁸ are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle said alkyl cycloalkyl, aryl, Het and heterocycle being optionally sustituted with R¹⁶⁰; e) NR¹¹¹R¹¹² wherein R¹¹¹ is H, (C₁₋₆)alkyl, or (C₃₋₇)cycloalkyl, and R¹¹² is H, (C₁₋₆)alkyl or (C₃₋₇cycloalkyl, COOR¹¹⁵ or SO₂R¹¹⁵ wherein R¹¹⁵ is (C₁₋₆)alkyl or (C₃₋₇)cycloalkyl, or both R¹¹¹ and R¹¹² are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl and heterocycle being optionally substituted with R¹⁶⁰; f) NR¹¹⁶COR¹¹⁷ wherein R¹¹⁶ and R¹¹⁷ is each H, (C₁₋₆)alkyl or (C₃₋₇)cycloalkyl said (C₁₋₆)alkyl and (C₃₋₇)cycloalkyl being optionally substituted with R¹⁶⁰; g) NR¹¹⁸CONR¹¹⁹R¹²⁰, wherein R¹¹⁸, R¹¹⁹ and R¹²⁰ is each H, (C₁₋₆)alkyl or (C₃₋₇)cycloalkyl, or R¹¹⁸ is covalently bonded to R¹¹⁹ and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle; or R¹¹⁹ and R¹²⁰ are covalently bonded together and to the nitrogen to which the are attached to form a 5, 6 or 7-membered saturated heterocycle; said alkyl, cycloalkyl, and heterocycle being optionally substituted with R¹⁶⁰; h) NR¹²¹COCOR¹²² wherein R¹²¹ is H, (C₁₋₆)alkyl or (₃₋₇)cycloalkyl, said alkyl and cycloalkyl being optionally substituted with R¹⁶⁰; or R¹²² is OR¹²³ or N(R¹²⁴)₂ wherein R¹²³ and each R¹²⁴ is independently H, (C₁₋₆alkyl) or (C₃₋₇)cycloalkyl, or both R¹²⁴ are covalently bonded together to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl and heterocycle being optionally substituted with R¹⁶⁰; i) COR¹²⁷ wherein R¹²⁷ is H, (C₁₋₆)alkyl or (C₃₋₇)cycloalkyl, said alkyl and cycloalkyl being optionally substituted with R¹⁶⁰; j) COOR¹²⁸ wherein R¹²⁸ is H, (C₁₋₆)alkyl or (C₃₋₇)cycloalkyl, said (C₁₋₆)alkyl and (C₃₋₇)cycloalkyl being optionally substituted with R¹⁶⁰; and k) CONR¹²⁹R¹³⁰ wherein R¹²⁹ and R¹³⁰ are independently H, (C₁₋₆)alkyl or (C₃₋₇)cycloalkyl or both R¹²⁹ and R¹³⁰ are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl and heterocycle being optionally substituted with R¹⁶⁰; wherein R¹⁶⁰ is defined as 1 or 2 substituents selected from: halogen CN, C₁₋₆alkyl, haloalkyl, COOR¹⁶¹, OR¹⁶¹, N(R¹⁶²)₂, SO₂N(R¹⁶²)₂, or CON(R¹⁶²)₂, wherein R¹⁶¹ and each R¹⁶² is independently H, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl; or both R¹⁶² are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle; R³ is selected from (C₃₋₇)cycloalkyl, (C₆₋₁₀)bicycloalkl, 6- or 10-membered aryl, or Het; R⁵ is —C(O)-Z, wherein Z is OR⁶ wherein R⁶ is C₁₋₆alkyl substituted with: 1 to 4 substituents selected from: OPO₃H, NO₂, cyano, azido, C(═NH)NH₂, C(═NH)NH(C₁₋₆)alkyl or C(═NH)NHCO(C₁₋₆)alkyl; or 1 to 4 substituents selected from: a) (C₁₋₆)alkyl or haloalkyl, (C₃₋₇)cycloalkyl, C₃₋₇spirocycloalkyl optionally containing 1 or 2 heteroatom, (C₂₋₆)alkenyl, (C₂₋₈)alkynyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, all of which optionally substituted with R¹⁵⁰; b) OR¹⁰⁴ wherein R¹⁰⁴ is (C₁₋₆alkyl) substituted with R¹⁵⁰, (C₃₋₇)cycloalkyl, or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, said cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het being optionally substituted with R¹⁵⁰; c) OCOR¹⁰⁵ wherein R¹⁰⁵ is (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, said alkyl, cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het being optionally substituted with R¹⁵⁰; d) SR¹⁰⁸, SO₃H, SO₂N(R¹⁰⁸)₂ or SO₂N(R¹⁰⁸)C(O)R¹⁰⁸ wherein each R¹⁰⁸ is independently H, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het or both R¹⁰⁸ are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het or heterocycle being optionally substituted with R¹⁵⁰; e) NR¹¹¹R¹¹² wherein R¹¹¹ is (C₁₋₆)alkyl substituted with R¹⁵⁰, (C₃₋₇)cycloakyl or (C₁₋₆)akyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, and R¹¹² is CN, (C₁₋₆)alkyl substituted with R¹⁵⁰, (C₃₋₇)cycloalkyl or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl, (C₁₋₆alkyl)aryl, Het, COOR¹¹⁵ or SO₂R¹¹⁵ wherein R¹¹⁵ is (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, or both R¹¹¹ and R¹¹² are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle, said cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, or heterocycle being optionally substituted with R¹⁵⁰; f) NR¹¹⁶COR¹¹⁷ wherein R¹¹⁶ and R¹¹⁷ is each H, (C₁₋₆)akyl, (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, said (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het being optionally substituted with R¹⁵⁰; g) NR¹¹⁸CONR¹¹⁹R¹²⁰, wherein R¹¹⁸, R¹¹⁹ and R¹²⁰ is each H, (C₁₋₆)alkyl, (C₃₋₇)cycloakyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, or R¹¹⁸ is covalently bonded to R¹¹⁹ and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle; or R¹¹⁹ and R¹²⁰ are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle; said alkyl, cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆)Het or heterocycle being optionally substituted with R¹⁵⁰; h) NR¹²¹COCOR¹²² wherein R¹²¹ and R¹²² is each is H, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, a 6- or 10-membered aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, said alkyl, cycloalkyl, alkyl-cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het being optionally substituted with R¹⁵⁰; or R¹²² is OR¹²³ or N (R¹²⁴)₂ wherein R¹²³and each R¹²⁴ is independently H, (C₁₋₆alkyl), (C₃₋₇)cycloalkyl, or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, or R¹²⁴ is OH or O(C₁₋₆alkyl) or both R¹²⁴ are covalently bonded together to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl, alkyl-cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or C₁₋₆alkyl)Het and heterocycle being optionally substituted with R¹⁵⁰; i) COR¹²⁷ wherein R¹²⁷ is H, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, said alkyl, cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆)Het being optionally substituted with R¹⁵⁰; j) COOR¹²⁸ wherein R¹²⁸ is (C₁₋₆)alkyl substituted with R¹⁵⁰, (C₃₋₇)cycloalkyl, or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, said (C₃₋₇)cycloalkyl or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl and (C₁₋₆alkyl)Het being optionally substituted with R¹⁵⁰; k) CONR¹²⁹R¹³⁰ wherein R¹²⁹ and R¹³⁰ are independently H, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, or both R¹²⁹ and R¹³⁰ are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl, alkyl-cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl, (C₁₋₆alkyl)Het and heterocycle being optionally substituted with R¹⁵⁰; l) aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, all of which being optionally substituted with R¹⁵⁰; wherein R¹⁵⁰ is: 1 to 3 substituents selected from: halogen, NO₂, cyano, azido or 1 to 3 substituents selected from: a) (C₁₋₆)alkyl or haloalkyl, (C₃₋₇)cycloalkyl, C₃₋₇spirocycloalkyl optionally containing 1 or 2 heteroatom, (C₂₋₆)alkenyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, all of which optionally substituted with R¹⁶⁰; b) OR¹⁰⁴ wherein R¹⁰⁴ is H, (C₁₋₆alkyl), (C₃₋₇)cycloalkyl, or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, said alkyl, cycloalkyl, aryl, Het, (C₁₋₆alkyl)laryl or (C₁₋₆alkyl)Het being optionally substituted with R¹⁶⁰; d) SO₃H, SO₂N(R¹⁰⁸)₂ or SO₂N(R¹⁰⁸)C(O)R¹⁰⁸ wherein each R¹⁰⁸ is independently H, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het or both R¹⁰⁸ are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het or heterocycle being optionally substituted with R¹⁶⁰; e) NR¹¹¹R¹¹² wherein R¹¹¹ is H, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, and R¹¹² is H, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl, Het, COOR¹¹⁵ or SO₂R¹¹⁵ wherein R¹¹⁵ is (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, or both R¹¹¹ and R¹¹² are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, or heterocycle being optionally substituted with R¹⁵⁰; f) NR¹¹⁶COR¹¹⁷ wherein R¹¹⁶ and R¹¹⁷ is each H, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, said (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het being optionally substituted with R¹⁶⁰; g) NR¹¹⁸CONR¹¹⁹R¹²⁰, wherein R¹¹⁸, R¹¹⁹ and R¹²⁰ is each H, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, or R¹¹⁹ and R¹²⁰ are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle; said alkyl, cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het or heterocycle being optionally substituted with R¹⁶⁰; h) NR¹²¹COCOR¹²² wherein R¹²³ is H, (C₁₋₆)alkyl optionally substituted with R¹⁶⁰; or R¹²² is OR¹²³ or N(R¹²⁴)₂ wherein R¹²³ and each R¹²⁴ is independently H, (C₁₋₆alkyl), (C₃₋₇)cycloalkyl, or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, or R¹²⁴ is OH or O(C₁₋₆alkyl) or both R¹²⁴ are covalently bonded together to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl, alkyl-cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het and heterocycle being optionally substituted with R¹⁶⁰; j) tetrazole, COOR¹²⁸ wherein R¹²⁸ is H, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, said (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl and (C₁₋₆alkyl)Het being optionally substituted with R¹⁶⁰; and k) CONR¹²⁹R¹³⁰ wherein R¹²⁹ and R¹³⁰ are independently H, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, or both R¹²⁹ and R¹³⁰ are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl, alkyl-cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl, (C₁₋₆alkyl)Het and heterocycle being optionally substituted with R¹⁶⁰; wherein R¹⁶⁰ is defined as 1 or 2 substituents selected from: tetrazole, halogen, CN, C₁₋₆alkyl, haloalkyl, COOR¹⁶¹, SO₃H, SO₂R¹⁶¹, OR¹⁶¹, N(R¹⁶²)₂, SO₂N(R¹⁶²)₂, or CON(R¹⁶²)₂, wherein R¹⁶¹ and each R¹⁶²is independently H, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl: or both R¹⁶² are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle; or Z is N(R^(6a))R⁶, wherein R^(6a) is H or (C₁₋₆alkyl) and R⁶ is (C₁₋₆)alkyl optionally substituted with: 1 to 4 substituents selected from: OPO₃H, NO₂, cyano, azido, C(═NH)NH₂, C(═NH)NH(C₁₋₆)alkyl or C(═NH)NHCO(C₁₋₆)alkyl; or 1 to 4 substituents selected from: a) (C₁₋₆)alkyl substituted with R^(150a), haloalkyl, (C₃₋₇)cycloalkyl, C₃₋₇ spirocycloalkyl optionally containing 1 or 2 heteroatom, (C₂₋₆)alkenyl, (C₂₋₈)alkynyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, all of which optionally substituted with R¹⁵⁰, wherein R^(150a) is the same as R¹⁵⁰ but is not halogen, OR^(150b), COOR^(150b), N(R^(150b))₂, wherein R^(150b) is H or C₁₋₆alkyl; b) OR¹⁰⁴ wherein R¹⁰⁴ is (C₁₋₆alkyl) substituted with R¹⁵⁰, (C₃₋₇)cycloalkyl, or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, said cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het being optionally substituted with R¹⁵⁰; c) OCOR¹⁰⁵ wherein R¹⁰⁵ is (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, said alkyl, cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het being optionally substituted with R¹⁵⁰; d) SO₃H, SO₂N(R¹⁰⁸)₂ or SO₂N(R¹⁰⁸)C(O)R¹⁰⁸ wherein each R¹⁰⁸ is independently H, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het or both R¹⁰⁸ are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het or heterocycle being optionally substituted with R¹⁵⁰; e) NR¹¹¹R¹¹² wherein R¹¹¹ is (C₁₋₆)alkyl substituted with R¹⁵⁰, (C₃₋₇)cycloalkyl or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, and R¹¹² is H, CN, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl, (C₁₋₆alkyl)Het or R¹¹¹ is H and R¹¹² is SO₂R¹¹⁵wherein R¹¹⁵ is (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, or both R¹¹¹ and R¹¹² are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle, said cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, or heterocycle being optionally substituted with R¹⁵⁰; f) NR¹¹⁶COR¹¹⁷ wherein R¹¹⁶ and R¹¹⁷ is each H, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, said (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het being optionally substituted with R¹⁵⁰; g) NR¹¹⁸CONR¹¹⁹R¹²⁰, wherein R¹¹⁸, R¹¹⁹ and R¹²⁰ is each H, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, or R¹¹⁸ is covalently bonded to R¹¹⁹ and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle; or R¹¹⁹ and R¹²⁰ are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle; said alkyl, cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het or heterocycle being optionally substituted with R¹⁵⁰; h) NR¹²¹COCOR¹²² wherein R¹²¹ and R¹²² is each is H, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, a 6- or 10-membered aryl, Het, (C₁₋₆)alkyl)aryl or (C₁₋₆alkyl)Het, said alkyl, cycloalkyl, alkyl-cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het being optionally substituted with R¹⁵⁰; or R¹²² is OR¹²³ or N(R¹²⁴)₂ wherein R¹²³ and each R¹²⁴ is independently H, (C₁₋₆alkyl), (C₃₋₇)cycloalkyl, or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, or R¹²⁴ is OH or O(C₁₋₆alkyl) or both R¹²⁴ are covalently bonded together to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl, alkyl-cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het and heterocycle being optionally substituted with R¹⁵⁰; i) COR¹²⁷ wherein R¹²⁷ is H, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, said alkyl, cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het being optionally substituted with R¹⁵⁰; j) COOR¹²⁸ wherein R¹²⁸ is (C₁₋₆)alkyl substituted with R¹⁵⁰, (C₃₋₇)cycloalkyl, or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, said (C₃₋₇)cycloalkyl, or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl and (C₁₋₆alkyl)Het being optionally substituted with R¹⁵⁰; k) CONR¹²⁹R¹³⁰ wherein R¹²⁹ and R¹³⁰ are independently H, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, or both R¹²⁹ and R¹³⁰ are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl, alkyl-cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl, (C₁₋₆alkyl)Het and heterocycle being optionally substituted with R¹⁵⁰; l) aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, all of which being optionally substituted with R¹⁵⁰; and wherein R¹⁵⁰ is selected from: 1 to 3 substituents selected from: halogen, NO₂, cyano, azido or 1 to 3 substituents selected from: a) (C₁₋₆)alkyl or haloalkyl, (C₃₋₇)cycloalkyl, C₃₋₇ spirocycloalkyl optionally containing 1 or 2 heteroatom, (C₂₋₆)alkenyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, all of which optionally substituted with R¹⁶⁰; b) OR¹⁰⁴ wherein R¹⁰⁴ is H, (C₁₋₆alkyl), (C₃₋₇)cycloalkyl, or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, said alkyl, cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het being optionally substituted with R¹⁶⁰; d) SO₃H, SO₂N(R¹⁰⁸)₂ or SO₂N(R¹⁰⁸)C(O)R¹⁰⁸ wherein each R¹⁰⁸ is independently H, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆-alkyl)aryl or (C₁₋₆alkyl)Het or both R¹⁰⁸ are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het or heterocycle being optionally substituted with R¹⁶⁰; e) NR¹¹¹R¹¹² wherein R¹¹¹ is H, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, and R¹¹² is H, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl, (C₁₋₆alkyl)Het, COOR¹¹⁵ or SO₂R¹¹⁵ wherein R¹¹⁵ is (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, or both R¹¹¹ and R¹¹² are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, or heterocycle being optionally substituted with R¹⁶⁰; f) NR¹¹⁶COR¹¹⁷ wherein R¹¹⁶ and R¹¹⁷ is each H, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, said (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het being optionally substituted with R¹⁶⁰; g) NR¹¹⁸CONR¹¹⁹R¹²⁰, wherein R¹¹⁸, R¹¹⁹ and R¹²⁰ is each H, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆-alkyl)Het, or R¹¹⁹ and R¹²⁰ are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle; said alkyl, cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het or heterocycle being optionally substituted with R¹⁶⁰; h) NR¹²¹COCOR¹²² wherein R¹²¹ is H, (C₁₋₆)alkyl optionally substituted with R¹⁶⁰; or R¹²² is OR¹²³ or N(R¹²⁴)₂ wherein R¹²³ and each R¹²⁴ is independently H, (C₁₋₆alkyl), (C₃₋₇)cycloalkyl, or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, or R¹²⁴ is OH or O(C₁₋₆alkyl) or both R¹²⁴ are covalently bonded together to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl, alkyl-cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het and heterocycle being optionally substituted with R¹⁶⁰; j) tetrazole, COOR¹²⁸ wherein R¹²⁸ is H, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, said (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl and (C₁₋₆alkyl)Het being optionally substituted with R¹⁶⁰; and k) CONR¹²⁹R¹³⁰ wherein R¹²⁹ and R¹³⁰ are independently H, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, or both R¹²⁹ and R¹³⁰ are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl, alkyl-cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl, (C₁₋₆alkyl)Het and heterocycle being optionally substituted with R¹⁶⁰; wherein R¹⁶⁰ is defined as 1 or 2 substituents selected from: tetrazole, halogen, CN, C₁₋₆alkyl, haloalkyl, COOR¹⁶¹, SO₃H, SO₂R¹⁶¹, OR¹⁶¹, N(R¹⁶²)₂, SO₂N(R¹⁶²)₂, or CON(R¹⁶²)₂, wherein R¹⁶¹ and each R¹⁶² is independently H, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl; or both R¹⁶² are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle; or R⁶ is

wherein, preferably, R⁷ and R⁸ are each independently H, (C₁₋₆)alkyl, haloalkyl, (C₃₋₇)cycloalkyl, 6- or 10-membered aryl, Het, (C₁₋₆)alkyl-aryl, (C₁₋₆)alkyl-Het, wherein said alkyl, cycloalkyl, aryl, Het, (C₁₋₆)alkyl-aryl, (C₁₋₆)alkyl-Het are optionally substituted with R⁷⁰; or R⁷ and R⁸ are covalently bonded together to form second (C₃₋₇)cycloalkyl or a 4, 5- or 6-membered heterocycle having from 1 to 3 heteroatom selected from O, N, and S; or when Z is N(R^(6a))R⁶, either of R⁷ or R⁸ is covalently bonded to R^(6a) to form a nitrogen-containing 5- or 6-membered heterocycle; wherein R⁷⁰ is selected from: 1 to 4 substituents selected from: halogen, NO₂, cyano, azido; or 1 to 4 substituents selected from: a) (C₁₋₆)alkyl or haloalkyl, (C₃₋₇)cycloalkyl, C₃₋₇ spirocycloalkyl optionally containing 1 or 2 heteroatom, (C₂₋₆)alkenyl, (C₂₋₈)alkynyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, all of which optionally substituted with R¹⁵⁰; b) OR¹⁰⁴ wherein R¹⁰⁴ is H, (C₁₋₆alkyl), (C₃₋₇)cycloalkyl, or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, said alkyl, cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het being optionally substituted with R¹⁵⁰; d) SO₂N(R¹⁰⁸)₂ or SO₂N(R¹⁰⁸)C(O)R¹⁰⁸ wherein each R¹⁰⁸ is independently H, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het or both R¹⁰⁸ are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het or heterocycle being optionally substituted with R¹⁵⁰; e) NR¹¹¹ R¹¹² wherein R¹¹¹ is H, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, and R¹¹² is H, CN, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl, (C₁₋₆alkyl)Het, COOR¹¹⁵ or SO₂R¹¹⁵wherein R¹¹⁵ is (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, or both R¹¹¹ and R¹¹² are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, or heterocycle being optionally substituted with R¹⁵⁰; f) NR¹¹⁶COR¹¹⁷ wherein R¹¹⁶ and R¹¹⁷ is each H, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, said (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆-alkyl)Het being optionally substituted with R¹⁵⁰; g) NR¹¹⁸CONR¹¹⁹R¹²⁰, wherein R¹¹⁸, R¹¹⁹ and R¹²⁰ is each H, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, or R¹¹⁸ is covalently bonded to R¹¹⁹ and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle; or R¹¹⁹ and R¹²⁰ are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle; said alkyl, cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het or heterocycle being optionally substituted with R¹⁵⁰; h) NR¹²¹COCOR¹²² wherein R¹²¹ is H, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, a 6- or 10-membered aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, said alkyl, cycloalkyl, alkyl-cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het being optionally substituted with R¹⁵⁰; and R¹²² is OR¹²³ or N(R¹²⁴)₂ wherein R¹²³ and each R¹²⁴ is independently H, (C₁₋₆alkyl), (C₃₋₇)cycloalkyl, or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, or R¹²⁴ is OH or O(C₁₋₆alkyl) or both R¹²⁴ are covalently bonded together to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl, alkyl-cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het and heterocycle being optionally substituted with R¹⁵⁰; i) COR¹²⁷ wherein R¹²⁷ is H, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, said alkyl, cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het being optionally substituted with R¹⁵⁰; j) COOR¹²⁸ wherein R¹²⁸ is H, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, said (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl and (C₁₋₆alkyl)Het being optionally substituted with R¹⁵⁰; k) CONR¹²⁹R¹³⁰ wherein R¹²⁹ and R¹³⁰ are independently H, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, or both R¹²⁹ and R¹³⁰ are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl, alkyl-cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl, (C₁₋₆alkyl)Het and heterocycle being optionally substituted with R¹⁵⁰; l) aryl, Het, (C₁₋₆alkyl)aryl or (C1-6alkyl)Het, all of which being optionally substituted with R¹⁵⁰; wherein R¹⁵⁰ is selected from: 1 to 3 substituents selected from: halogen, NO₂, cyano, azido; or 1 to 3 substituents selected from: a) (C₁₋₆)alkyl or haloalkyl, (C₃₋₇)cycloalkyl, C₃₋₇ spirocycloalkyl optionally containing 1 or 2 heteroatom, (C₂₋₆)alkenyl, (C₂₋₈)alkynyl, all of which optionally substituted with R¹⁶⁰; b) OR¹⁰⁴ wherein R¹⁰⁴ is H, (C₁₋₆alkyl) or (C₃₋₇)cycloalkyl, said alkyl and cycloalkyl being optionally substituted with R¹⁶⁰; d) SO₂N(R¹⁰⁸)₂ wherein R¹⁰⁸ is H, (C₁₋₆)alkyl or (C₃₋₇)cycloalkyl, said alkyl or cycloalkyl being optionally substituted with R¹⁶⁰; e) NR¹¹¹ R¹¹² wherein R¹¹¹ is H, (C₁₋₆)alkyl or (C₃₋₇)cycloalkyl, and R¹¹² is H, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl, (C₁₋₆alkyl)Het, COOR¹¹⁵ or SO₂R¹¹⁵ wherein R¹¹⁵ is (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, or both R¹¹¹ and R¹¹² are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, or heterocycle being optionally substituted with R¹⁶⁰; f) NR¹¹⁶COR¹¹⁷ wherein R¹¹⁶ and R¹¹⁷ is each H, (C₁₋₆)alkyl or (C₃₋₇)cycloalkyl, said (C₁₋₆)alkyl or (C₃₋₇)cycloalkyl being optionally substituted with R¹⁶⁰; g) NR¹¹⁸CONR¹¹⁹R¹²⁰, wherein R¹¹⁸, R¹¹⁹ and R¹²⁰ is each H, (C₁₋₆)alkyl or (C₃₋₇)cycloalkyl; or R¹¹⁹ and R¹²⁰ are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle; said alkyl, cycloalkyl or heterocycle being optionally substituted with R¹⁶⁰; h) NR¹²¹COCOR¹²² wherein R¹²¹ is H, (C₁₋₆)alkyl or (C₃₋₇)cycloalkyl, said alkyl or cycloalkyl being optionally substituted with R¹⁶⁰; or R¹²² is OR¹²³ or N(R¹²⁴)₂ wherein R¹²³ and each R¹²⁴ is independently H, (C₁₋₆alkyl) or (C₃₋₇)cycloalkyl, or R¹²⁴ is OH or O(C₁₋₆alkyl) or both R¹²⁴ are covalently bonded together to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl and heterocycle being optionally substituted with R¹⁶⁰; j) tetrazole, COOR¹²⁸ wherein R¹²⁸ is H, (C₁₋₆)alkyl or (C₃₋₇)cycloalkyl, said (C₁₋₆)alkyl and (C₃₋₇)cycloalkyl being optionally substituted with R¹⁶⁰; and k) CONR¹²⁹R¹³⁰ wherein R¹²⁹ and R¹³⁰ are independently H, (C₁₋₆)alkyl or (C₃₋₇)cycloalkyl, or both R¹²⁹ and R¹³⁰ are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl and heterocycle being optionally substituted with R¹⁶⁰; wherein R¹⁶⁰ is defined as 1 or 2 substituents selected from: tetrazole, halogen, CN, C₁₋₆alkyl, haloalkyl, COOR¹⁶¹, OR¹⁶¹, N(R¹⁶²)₂ or CON(R¹⁶²)₂, wherein R¹⁶¹ and each R¹⁶² is independently H or (C₁₋₆)alkyl; Y² is O or S; R⁹ is H; or R⁹ is covalently bonded to either of R⁷ or R⁸ to form a 5- or 6-membered heterocycle; and Q is a 6- or 10-membered aryl, Het, all of which being optionally substituted with:

wherein R¹⁰⁰is: 1 to 4 substituents selected from: halogen, NO₂, cyano or azido; or 1 to 4 substituents selected from: a) (C₁₋₆)alkyl or haloalkyl, (C₃₋₇)cycloalkyl, (C₂₋₆)alkenyl, (C₂₋₈)alkynyl, (C₁₋₆) alkyl-(C₃₋₇)cycloalkyl, all of which optionally substituted with R¹⁵⁰; b) OR¹⁰⁴ wherein R¹⁰⁴ is H, (C₁₋₆alkyl), (C₃₋₇)cycloalkyl, or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, said alkyl, cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het being optionally substituted with R¹⁵⁰; e) NR¹¹¹R¹¹² wherein R¹¹¹ is H, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, and R¹¹² is H, CN, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl, (C₁₋₆alkyl)Het, COOR¹¹⁵ or SO₂R¹¹⁵wherein R¹¹⁵ is (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, or both R¹¹¹ and R¹¹² are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, or heterocycle being optionally substituted with R¹⁵⁰; f) NR¹¹⁶COR¹¹⁷ wherein R¹¹⁶ and R¹¹⁷ is each H, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, said (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het being optionally substituted with R¹⁵⁰; g) NR¹¹⁸CONR¹¹⁹R¹²⁰, wherein R¹¹⁸, R¹¹⁹ and R¹²⁰ is each H, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, or R¹¹⁸ is covalently bonded to R¹¹⁹ and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle; or R¹¹⁹ and R¹²⁰ are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle; said alkyl, cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het or heterocycle being optionally substituted with R¹⁵⁰; h) NR¹²¹COCOR¹²² wherein R¹²¹ and R¹²² is each is H, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, a 6- or 10-membered aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, said alkyl, cycloalkyl, alkyl-cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het being optionally substituted with R¹⁵⁰; or R¹²² is OR¹²³ or N(R¹²⁴)₂ wherein R¹²³ and each R¹²⁴ is independently H, (C₁₋₆alkyl), (C₃₋₇)cycloalkyl, or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, or R¹²⁴ is OH or O(C₁₋₆alkyl) or both R¹²⁴ are covalently bonded together to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl, alkyl-cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het and heterocycle being optionally substituted with R¹⁵⁰; j) COOR¹²⁸ wherein R¹²⁸ is H, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, said (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl and (C₁₋₆alkyl)Het being optionally substituted with R¹⁵⁰; k) CONR¹²⁹R¹³⁰ wherein R¹²⁹ and R¹³⁰ are independently H, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, or both R¹²⁹ and R¹³⁰ are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl, alkyl-cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl, (C₁₋₆alkyl)Het and heterocycle being optionally substituted with R¹⁵⁰; l) aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, all of which being optionally substituted with R¹⁵⁰; wherein R¹⁵⁰ is selected from: 1 to 3 substituents selected from: halogen, NO₂, cyano or azido; or 1 to 3 substituents selected from: a) (C₁₋₆)alkyl or haloalkyl, (C₃₋₇)cycloalkyl, C₃₋₇ spirocycloalkyl optionally containing 1 or 2 heteroatom, (C₂₋₆)alkenyl, (C₂₋₈)alkynyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, all of which optionally substituted with R¹⁶⁰; b) OR¹⁰⁴ wherein R¹⁰⁴ is H, (C₁₋₆alkyl), (C₃₋₇)cycloalkyl, or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, said alkyl, cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het being optionally substituted with R¹⁶⁰; d) SO₃H, SO₂N(R¹⁰⁸)₂ or SO₂N(R¹⁰⁸)C(O)R¹⁰⁸ wherein each R¹⁰⁸ is independently H, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het or both R¹⁰⁸ are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het or heterocycle being optionally substituted with R¹⁶⁰; e) NR¹¹¹R¹¹² wherein R¹¹¹ is H, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, and R¹¹² is H, CN, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl, (C₁₋₆alkyl)Het or SO₂R¹¹⁵ wherein R¹¹⁵ is (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, or both R¹¹¹ and R¹¹² are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, or heterocycle being optionally substituted with R¹⁶⁰; f) NR¹¹⁶COR¹¹⁷ wherein R¹¹⁶ and R¹¹⁷ is each H, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, said (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het being optionally substituted with R¹⁶⁰; g) NR¹¹⁸CONR¹¹⁹R¹²⁰, wherein R¹¹⁸, R¹¹⁹ and R¹²⁰ is each H, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, or R¹¹⁸ is covalently bonded to R¹¹⁹ and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle; or R¹¹⁹ and R¹²⁰ are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle; said alkyl, cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het or heterocycle being optionally substituted with R¹⁶⁰; h) NR¹²¹COCOR¹²² wherein R¹²¹ is H, (C₁₋₆)alkyl optionally substituted with R¹⁶⁰; or R¹²² is OR¹²³ or N(R¹²⁴)₂ wherein R¹²³ and each R¹²⁴ is independently H, (C₁₋₆alkyl), (C₃₋₇)cycloalkyl, or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, or R¹²⁴ is OH or O(C₁₋₆alkyl) or both R¹²⁴ are covalently bonded together to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl, alkyl-cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het and heterocycle being optionally substituted with R¹⁶⁰; j) tetrazole, COOR¹²⁸ wherein R¹²⁸ is H, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, said (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl and (C₁₋₆alkyl)Het being optionally substituted with R¹⁶⁰; and k) CONR¹²⁹R¹³⁰ wherein R¹²⁹ and R¹³⁰ are independently H, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, or both R¹²⁹ and R¹³⁰ are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl, alkyl-cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl, (C₁₋₆alkyl)Het and heterocycle being optionally substituted with R¹⁶⁰; wherein R¹⁶⁰ is defined as 1 or 2 substituents selected from: tetrazole, halogen, CN, C₁₋₆alkyl, haloalkyl, COOR¹⁶¹, SO₃H, SR¹⁶¹, SO₂R¹⁶¹, OR¹⁶¹, N(R¹⁶²)₂, SO₂N(R¹⁶²)₂, or CON(R¹⁶²)₂, wherein R¹⁶¹ and each R¹⁶² is independently H, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl; or both R¹⁶² are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle; or an enantiomer, diastereomer or tautomer thereof, including a salt thereof; wherein said probe comprises a detectable label attached to any suitable position, whereby said probe binds to an HCV polymerase and is capable of being displaced by an inhibitor thereof and whereby a modulation in said signal is an indication that said test compound binds to said polymerase.
 2. The method according to claim 1 wherein the detectable label is selected from the group consisting of: a fluorescent label, a radioactive atom, a chemiluminescent label, and a colorimetric label.
 3. The method according to claim 2 wherein said label is a fluorescent label or chemiluminescent label.
 4. The method according to claim 3, wherein the fluorescent label is selected from the group consisting of: fluorescein, Oregon green, dansyl, rhodamine, Texas-red, phycoerythrin and Eu⁺.
 5. The method according to claim 4, wherein the fluorescent label is fluorescein.
 6. The method according to claim 2, wherein the radioactive atom is selected from the group consisting of ³H, ¹⁴C and ¹²⁵I.
 7. The method according to claim 1, wherein the detectable label is a fluorescent reporter/quencher pair.
 8. The method according to claim 7, wherein the reporter/quencher pair is selected from the group consisting of: EDANS/DABCYL, tryptophan/2,4-dinitrophenyl, tryptophan/DANSYL, 7-methoxycoumarin/2,4-dinitrophenyl, 2-aminobenzoyl/2,4-dinitrophenyl and 2-aminobenzoyl/3-nitrotyrosine.
 9. The method according to claim 1, wherein the probe is selected from:


10. A method for development of an assay for identifying inhibitors of Hepatitis C Virus polymerase said method comprising using a probe of formula I, according to claim 1, in the development of an assay for identifying inhibitors of HCV polymerase.
 11. A method for identifying compounds that inhibit Hepatitis C Virus polymerase comprising the steps of: a) contacting an Hepatitis C Virus polymerase with a probe of formula la, according to claim 1, so as to form a complex having said probe bound to said polymerase; b) measuring the signal emitted from said probe in said complex to establish a base line signal; c) incubating 4complex from step a) with a test compound; and d) measuring said signal from said complex from step C): and e) comparing said signal from step d) with said signal from step b); whereby a modulation in said signal is an indication that said test compound inhibits said polymerase.
 12. A method for identifying compounds capable of inhibiting Hepatitis C Virus polymerase, comprising: f) repeating steps (a) to (e), according to claim 11 in a high throughput screen.
 13. The method according to claim 11, wherein the Hepatitis C Virus polymerase is selected from the group consisting of NS5B; NSSBΔ21: and NS5BΔ57.
 14. The method according to claim 11, wherein the Hepatitis C virus polymerase is obtained from genotype HCV-1a or HCV-1b strains optionally having a histidine tag at either the N- or C-terminal.
 15. The method according to claim 1, wherein said probe is a compound having the following formula:

wherein R³ is (C₅₋₆)cycloalkyl; R² is phenyl, or Het both being optionally substituted with R²⁰; R⁷, R⁸, R⁹, R¹⁰⁰, and R¹⁵⁰ are as defined according to claim 1; R¹¹ is OPO₃H, NO₂, cyano, azido, C(═NH)NH₂, C(═NH)NH(C₁₋₆)alkyl or C(═NH)NHCO(C¹⁻⁶)alkyl; or a) (C¹⁻⁶)alkyl substituted with R^(150a), haloalkyl, (C₃₋₇)cycloalkyl, C₃₋₇ spirocycloalkyl optionally containing 1 or 2 heteroatom, (C₂₋₆)alkenyl, (C₂₋₆)alkynyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, all of which optionally substituted with R¹⁵⁰, wherein R^(150a) is the same as R¹⁵⁰ but is not halogen, OR^(150b), COOR^(150b), N(R^(150b))₂, wherein R^(150b) is H or C₁₋₆alkyl; b) OR¹⁰⁴ wherein R¹⁰⁴ is (C₁₋₆alkyl) substituted with R¹⁵⁰, (C₃₋₇)cycloalkyl, or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, said cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het being optionally substituted with R¹⁵⁰; c) OCOR¹⁰⁵ wherein R¹⁰⁵ is (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, said alkyl, cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het being optionally substituted with R¹⁵⁰; d) SO₃H, SO₂N(R¹⁰⁸)₂ or SO₂N(R₁₀₈)C(O)R¹⁰⁸ wherein each R¹⁰⁸ is independently H, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het or both R¹⁰⁸ are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl, aryl, Het, (C₁₋₆)alkyl)aryl or (C₁₋₆alkyl)Het or heterocycle being optionally substituted with R¹⁵⁰; e) NR¹¹¹R¹¹² wherein R¹¹¹ is (C₁₋₆)alkyl substituted with R¹⁵⁰, (C₃₋₇)cycloalkyl or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, and R¹¹² is H, CN, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl, (C₁₋₆alkyl)Het or R¹¹¹ is H and R¹¹² is SO₂R¹¹⁵ wherein R¹¹⁵ is (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, or both R¹¹¹ and R¹¹² are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or Tmembered saturated heterocycle, said cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, or heterocycle being optionally substituted with R¹⁵⁰; f) NR¹¹⁸R¹¹⁷ wherein R¹¹⁸ and R¹¹⁷ is each H, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, (C₁₋₆alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, said (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl, (C₁₋₆alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het being optionally substituted with R¹⁵⁰; g) NR¹¹⁸CONR¹¹⁰R¹²⁰, wherein R¹¹⁸, R¹¹⁹ and R¹²⁰ is each H, (C₁₋₆)alkyl, (C₁₋₆)alkyl, C₃₋₇)cycloalkyl, (C₁₋₆)alkyl-(C₁₋₆)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, or R¹¹⁸ is covalently bonded to R¹¹⁹ and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle; or R¹¹⁹ and R¹²⁰ are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle; said alkyl, cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het or heterocycle being optionally substituted with R¹⁵⁰; h) NR¹²¹COCOR¹²² wherein R¹²¹ and R₁₂₂ is each is H, (C₁₋₆)alkyl, (C³⁻⁷)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, a 6- or 10-membered aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, said alkyl, cycloalkyl, alkyl-cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het being optionally substituted with R¹⁵⁰; or R¹²² is OR¹²³ or N(R¹²³)₂ wherein R¹²³ and each R¹²⁴ is independently H, (C₁₋₆alkyl), (C₃₋₇)cycloalkyl, or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, or R¹²⁴ is OH or O(C₁₋₆alkyl) or both R¹²⁴ are covalently bonded together to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl, alkyl-cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het and heterocycle being optionally substituted with R¹⁵⁰; i) COR¹²⁷ wherein R¹²⁷ is H, (C₁₋₆)alkyl, (C₃₋₇)cycloalkyl or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, said alkyl, cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het being optionally substituted with R¹⁵⁰; j) COOR¹²⁸ wherein R¹²⁸ is H or (C₁₋₆)alkyl substituted with R¹⁵⁰, (C₃₋₇)cycloalkyl, or (C₁₋₆)alkyl-(C₃₋₇cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, said (C₃₋₇)cycloalkyl, or (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl and (C₁₋₆alkyl)Het being optionally substituted with R¹⁵⁰; k) CONR¹²⁹R¹³⁰ wherein R¹²⁹ and R¹³⁰ are independently H, (C₁₋₆alkyl, (C³⁻⁷)cycloalkyl, (C₁₋₆)alkyl-(C₃₋₇)cycloalkyl, aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, or both R¹²⁹ and R¹³⁰ are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl, alkyl-cycloalkyl, aryl, Het, (C¹⁻⁶alkyl)aryl, (C₁₋₆alkyl)Het and heterocycle being optionally substituted with R¹⁵⁰; l) aryl, Het, (C₁₋₆alkyl)aryl or (C₁₋₆alkyl)Het, all of which being optionally substituted with R¹⁵⁰ wherein R¹⁵⁰ is as defined in claim 1; or a salt thereof; wherein said compound is optionally: a) marked with a radioactive isotope at any suitable position, b) linked to a detectable moiety by a suitable linker at any suitable position, except R³; or c) linked to an affinity tag at any suitable position, except R³.
 16. The method according to claim 1, wherein said probe is a compound having the following formula:

wherein R³ is (C₅₋₆)cycloalkyl; R² is phenyl, or Het both being optionally substituted with R²⁰; R¹⁵⁰ are as defined in claim 1; or a salt thereof: wherein said compound is optionally: a) marked with a radioactive isotope at any suitable position; b) linked to a detectable moiety by a suitable linker at any suitable position, except R³; or c) linked to an affinity tag at any suitable position, except R³. 